High affinity pd-1 agents and methods of use

ABSTRACT

High affinity PD-1 mimic polypeptides are provided, which (i) comprise at least one amino acid change relative to a wild-type PD-1 protein; and (ii) have an increased affinity for PD-L1 relative to the wild-type protein. Compositions and methods are provided for modulating the activity of immune cells in a mammal by administering a therapeutic dose of a pharmaceutical composition comprising a high affinity PD-1 mimic polypeptide, which blocks the physiological binding interaction between PD-1 and its ligand PD-L1 and/or PD-L2.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication Nos. 62/035,316 filed Aug. 8, 2014, and 62/150,789, filedApr. 21, 2015, each of which applications is incorporated herein byreference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file,“STAN-1136US2_SeqList_ST25.txt” created on Aug. 7, 2015 and having asize of 119 KB. The contents of the text file are incorporated byreference herein in their entirety.

INTRODUCTION

T cell activation depends on an antigen specific signal provided to Tcell receptors. Additional signals, for example costimulatory (positive)and/or coinhibitory (negative) signals, fine-tune this response, helpingto determine its strength, nature, and duration. Costimulatoryinteractions potentiate the activation and proliferation of T cells,while coinhibitory interactions promote regulation. For example, CD28and CTLA-4 coreceptors both bind to the B7-1 (CD80) and B7-2 (CD86)molecules. CD28 acts as a strong positive costimulatory receptor andCTLA-4 as a potent coinhibitory receptor.

A receptor known as the programmed death-1 (PD-1) receptor is expressedon T cells, B cells and myeloid cells, and binds to the programmed deathligand (PD-L). This receptor-ligand pair functions primarily to provideinhibitory signals (e.g., through the recruitment of phosphatases, suchas SHP-2, to the immunoreceptor tyrosin-based switch motif (ITSM) of thecytoplasmic tail of PD-1).

PD-1 signaling plays an important role in inducing and maintainingperipheral tolerance. PD-1 ligands (PD-Ls) on antigen presenting cellshave been shown to inhibit autoreactive T cells and induce peripheraltolerance, whereas those on parenchymal cells prevent tissue destructionby suppressing effector T cells to maintain tolerance. The inhibitoryrole of PD-1 is highlighted by the phenotype of PD-1 deficient mice,which develop various autoimmune diseases, depending on the geneticbackground. The PD-1/PD-L pathway is frequently exploited as a targetfor immune evasion by tumor cells and by a wide range of pathogens Forexample, the PD1:PD-L pathway can be exploited (e.g., hyperactivated) bytumors and viruses (e.g., viruses that cause chronic infection), whichcan express PD-L proteins to stimulate PD-1 (e.g., on T-cells), therebyreducing immune cell (e.g., T-cell) responses and evading eradication bythe immune system.

The present disclosure provides high affinity PD-1 mimic polypeptidesthat mimic PD-1 by specifically binding to PD-L1, blocking/reducing theinteraction of PD-L1 with PD-1 on the surface of cells (e.g., immunecells such as T cells), and thereby blocking/reducing PD-L1 stimulatedPD-1 activity. Also disclosed are methods of using high affinity PD-1mimic polypeptides to reduce PD-1 activity.

SUMMARY

High affinity PD-1 variant (mimic) polypeptides are provided. Thepolypeptides are sequence variants of a wild type PD-1 protein (e.g.,the wild type human PD-1 protein), and have utility for in vivo and invitro methods that block the interaction between a wild type PD-1protein and its ligand PD-L (PD-L1 and/or PD-L2). A high affinity PD-1mimic polypeptide includes at least one amino acid change relative to awild-type PD-1 protein, has an increased affinity for PD-L (PD-L1 and/orPD-L2) relative to the wild-type PD-1 protein, and lacks a transmembranedomain of a wild type PD-1 protein. The amino acid changes that providefor increased affinity can be localized to amino acid positions ofcontact between PD-1 and PD-L, and/or can be located in theimmunoglobulin domain of PD-1 protein from which it was derived.

A high affinity PD-1 mimic polypeptide can be post-translationallymodified, for example by glycosylation, PEGylation, etc. A high affinityPD-1 mimic polypeptide can be a fusion protein (i.e., can includeadditional amino acid sequences), for example a fusion with antibody Fcsequences and/or a variable region of an antibody that provides forspecific binding to an antigen of interest; and the like. High affinityPD-1 mimic polypeptides may be monomeric or multimeric, i.e. dimer,trimer, tetramer, etc.

In some embodiments, methods are provided for modulating the activity ofimmune cells (T cells, NK cells, etc.) in a mammal by administering atherapeutic dose of a pharmaceutical composition comprising a highaffinity PD-1 mimic polypeptide, which blocks the physiological bindinginteraction between PD-1 and its ligand PD-L1 and/or PD-L2.

The disclosure also includes pharmaceutical formulations having a highaffinity PD-1 mimic polypeptide in combination with a pharmaceuticallyacceptable excipient. Such formulations may be provided as a unit dose,e.g., a dose effective to block the interaction of PD-1 on a first cellwith PD-L (PD-L1 and/or PD-L2) on a second cell within an individual.Pharmaceutical formulations also include lyophilized or otherpreparations of the high affinity PD-1 mimic polypeptides, which may bereconstituted for use.

In some embodiments, methods are provided to stimulate an immuneresponse towards target cells, e.g., targeting the destruction of livingcancer cells by the immune system. In such methods, a cell expressingPD-L1 is contacted with a high affinity PD-1 mimic polypeptide in a doseeffective to block the interaction between endogenous PD-1 (e.g., on afirst cell) and PD-L (PD-L1 and/or PD-L2, e.g., on a second cell).Blocking this interaction allows the immune-based destruction of targetcells that are not destroyed in the absence of the high affinity PD-1mimic polypeptide. The contacting may be performed in vivo, e.g., fortherapeutic purposes, and in vitro, e.g., for screening assays and thelike. The high affinity PD-1 mimic polypeptide for these purposes may bemultimeric; or monomeric. Monomeric reagents find particular use foradministration in combination with an antibody that selectively binds tothe targeted cell.

Inflicted individuals that can be treated with a high affinity PD-1mimic polypeptide include individuals that have cancer, individuals thatharbor an infection (e.g., a chronic infection, a viral infection,etc.), individuals that have an immunological disorder (e.g., a disorderassociated with immunosuppression), individuals that have aninflammatory disorder, and/or individuals that have otherhyper-proliferative conditions, for example sclerosis, fibrosis, and thelike, etc. In some cases, cancer cells, e.g., tumor cells, are targetedfor elimination by contacting the cells of the immune system with a doseof a high affinity PD-1 mimic polypeptide that is effective to block, ormask the interaction of PD-1 with PD-L, allowing for increasedstimulation of the immune system. In some cases, the targeted cell(e.g., an inflicted cell such as a cancer cell, a tumor cell, aninfected cell, etc.) expresses PD-L1 and/or PD-L2, and a high affinityPD-1 mimic polypeptide blocks the interaction of PD-L on the targetedcell with PD-1 on an immune cell (e.g., a T cell, an NK cell, etc.),which can block the ability of the targeted cell to suppress an immuneresponse against the targeted cell.

Administration of an effective dose of high affinity PD-1 mimicpolypeptide to a patient prevents interaction between PD-1 and PD-L1,which can increase the clearance of tumor cells and/or infected cells(e.g., chronically infected cells). In some cases, the high affinityPD-1 mimic polypeptide can be combined with monoclonal antibodiesdirected against one or more tumor cell markers, which combinationtherapy can be synergistic in enhancing elimination of cancer cells ascompared to the administration of either agent as a single entity. Inother embodiments the high affinity PD-1 mimic polypeptide comprises adetectable label. Such a labeled reagent can be used for imagingpurposes in vitro or in vivo, e.g., in the imaging of a tumor. In somecases, a high affinity PD-1 mimic polypeptide can be used as adiagnostic tool for the detection of PD-L (e.g, cells expressing PD-L1),and can be used as a companion diagnostic to assess whether a particulartreatment regimen has been successful.

Provided are high affinity PD-1 mimic polypeptides. In some cases, ahigh affinity PD-1 mimic polypeptide is a variant of a wild-type PD-1sequence, but lacks the PD-1 transmembrane domain, and comprises one ormore amino acid changes relative to a corresponding sequence of the wildtype PD-1 polypeptide, where the one or more amino acid changesincreases the affinity of the polypeptide for PD-L1 as compared to theaffinity for PD-L1 of the corresponding wild type PD-1 polypeptide. Insome cases, the PD-1 mimic polypeptide has a K_(d) of 1×10⁻⁷ M or lessfor PD-L1. In some cases, the affinity for PD-L1 of the high affinityPD-1 mimic polypeptide is 5-fold or more greater than the affinity forPD-L1 of said PD-1 mimic polypeptide that does not have an amino acidchange relative to a corresponding sequence of a wild type PD-1polypeptide. In some cases, the high affinity PD-1 mimic polypeptide hasa decreased affinity for PD-L2 as compared to the affinity for PD-L2 ofsaid PD-1 mimic polypeptide that does not have an amino acid changerelative to a corresponding sequence of a wild type PD-1 polypeptide. Insome cases, the one or more amino acid changes is located at an aminoacid position of PD-1 that contacts PD-L1. In some cases, the one ormore amino acid changes is located at an amino acid position, relativeto the protein fragment set forth in SEQ ID NO: 2, selected from: V39,N41, Y43, M45, S48, N49, Q50, T51, D52, K53, A56, Q63, G65, Q66, L97,S102, L103, A104, P105, K106, and A107; or the corresponding amino acidposition relative to another wild type PD-1 protein. In some cases, theone or more amino acid changes is located at an amino acid position,relative to the protein fragment set forth in SEQ ID NO: 2, selectedfrom: V39, L40, N41, Y43, R44, M45, S48, N49, Q50, T51, D52, K53, A56,Q63, G65, Q66, V72, H82, M83, R90, Y96, L97, A100, S102, L103, A104,P105, K106, and A107; or the corresponding amino acid position relativeto another wild type PD-1 protein. In some cases, the one or more aminoacid changes is 5 or more amino acid changes.

Also provided are high affinity PD-1 mimic polypeptides comprising oneor more amino acid changes, relative to the PD-1 protein fragment setforth in SEQ ID NO: 2, selected from: (1) V39H or V39R; (2) L40V orL40I; (3) N41I or N41V; (4) Y43F or Y43H; (5) R44Y or R44L; (6) M45Q,M45E, M45L, or M45D; (7) S48D, S48L, S48N, S48G, or S48V; (8) N49C,N49G, N49Y, or N49S; (9) Q50K, Q50E, or Q50H; (10) T51V, T51L, or T51A;(11) D52F, D52R, D52Y, or D52V; (12) K53T or K53L; (13) A56S or A56L;(14) Q63T, Q63I, Q63E, Q63L, or Q63P; (15) G65N, G65R, G65I, G65L, G65F,or G65V; (16) Q66P; (17) V72I; (18) H82Q; (19) M83L or M83F; (20) R90K;(21) Y96F; (22) L97Y, L97V, or L97I; (23) A100I or A100V; (24) S102T orS102A; (25) L103I, L103Y, or L103F; (26) A104S, A104H, or A104D; (27)P105A; (28) K106G, K106E, K106I, K106V, K106R, or K106T; and (29) A107P,A107I, or A107V; or a change that results in the same amino acid at thecorresponding position relative to another wild type PD-1 protein.

Also provided are high affinity PD-1 mimic polypeptides comprising aminoacid changes located at amino acid positions, relative to the proteinfragment set forth in SEQ ID NO: 2, selected from: (a) V39, N41, Y43,M45, S48, N49, Q50, K53, A56, Q63, G65, Q66, L97, S102, L103, A104,K106, and A107, or the corresponding amino acid positions relative toanother wild type PD-1 protein; (b) V39, N41, Y43, M45, S48, Q50, T51,D52F, K53, A56, Q63, G65, Q66, L97, S102, L103, A104, K106, and A107, orthe corresponding amino acid positions relative to another wild typePD-1 protein; (c) V39, L40, N41, Y43, R44, M45, N49, K53, M83, L97,A100, and A107, or the corresponding amino acid positions relative toanother wild type PD-1 protein; (d) V39, L40, N41, Y43, M45, N49, K53,Q66P, M83, L97, and A107, or the corresponding amino acid positionsrelative to another wild type PD-1 protein; (e) V39, L40, N41, Y43, M45,N49, K53, Q66P, H82, M83, L97, A100, and A107, or the correspondingamino acid positions relative to another wild type PD-1 protein; (f)V39, L40, N41, Y43, M45, N49, K53, M83, L97, A100, and A107, or thecorresponding amino acid positions relative to another wild type PD-1protein; (g) V39, L40, N41, Y43, R44, M45, N49, K53, L97, A100, andA107, or the corresponding amino acid positions relative to another wildtype PD-1 protein; and (h) V39, L40, N41, Y43, M45, N49, K53, L97, A100,and A107, or the corresponding amino acid positions relative to anotherwild type PD-1 protein.

Also provided are high affinity PD-1 mimic polypeptides comprising aminoacid changes, relative to the PD-1 protein fragment set forth in SEQ IDNO: 2, selected from: (a) {V39H or V39R}, {N41I or N41V}, {Y43F orY43H}, {M45Q, M45E, M45L, or M45D}, {548D, S48L, S48N, S48G, or S48V},{N49C, N49G, N49Y, or N49S}, {Q50K, Q50E, or Q50H}, {K53T or K53L},{A56S or A56L}, {Q63T, Q63I, Q63E, Q63L, or Q63P}, {G65N, G65R, G65I,G65L, G65F, or G65V}, {Q66P}, {L97Y, L97V, or L97I}, {S102T or S102A},{L103I, L103Y, or L103F}, {A104S, A104H, or A104D}, {K106G, K106E,K106I, K106V, K106R, or K106T}, and {A107P, A107I, or A107V}; or changesthat result in the same amino acids at the corresponding positionsrelative to another wild type PD-1 protein; (b) {V39H or V39R}, {N41I orN41V}, {Y43F or Y43H}, {M45Q, M45E, M45L, or M45D}, {548D, S48L, S48N,S48G, or S48V}, {Q50K, Q50E, or Q50H}, {T51V, T51L, or T51A}, {D52F,D52R, D52Y, or D52V}, {K53T or K53L}, {A56S or A56L}, {Q63T, Q63I, Q63E,Q63L, or Q63P}, {G65N, G65R, G65I, G65L, G65F, or G65V}, {Q66P}, {L97Y,L97V, or L97I}, {S102T or S102A}, {L103I, L103Y, or L103F}, {A104S,A104H, or A104D}, {K106G, K106E, K106I, K106V, K106R, or K106T}, and{A107P, A107I, or A107V}; or changes that result in the same amino acidsat the corresponding positions relative to another wild type PD-1protein; (c) {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43F orY43H}, {R44Y or R44L}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y,or N49S}, {K53T or K53L}, {M83L or M83F}, {L97Y, L97V, or L97I}, {A100Ior A100V}, and {A107P, A107I, or A107V}; or changes that result in thesame amino acids at the corresponding positions relative to another wildtype PD-1 protein; (d) {V39H or V39R}, {L40V or L40I}, {N41I or N41V},{Y43F or Y43H}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, orN49S}, {K53T or K53L}, {Q66P}, {M83L or M83F}, {L97Y, L97V, or L97I},and {A107P, A107I, or A107V}; or changes that result in the same aminoacids at the corresponding positions relative to another wild type PD-1protein; (e) {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43F orY43H}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, or N49S}, {K53Tor K53L}, {Q66P}, {H82Q}, {M83L or M83F}, {L97Y, L97V, or L97I}, {A100Ior A100V}, and {A107P, A107I, or A107V}; or changes that result in thesame amino acids at the corresponding positions relative to another wildtype PD-1 protein; (f) {V39H or V39R}, {L40V or L40I}, {N41I or N41V},{Y43F or Y43H}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, orN49S}, {K53T or K53L}, {M83L or M83F}, {L97Y, L97V, or L97I}, {A100I orA100V}, and {A107P, A107I, or A107V}; or changes that result in the sameamino acids at the corresponding positions relative to another wild typePD-1 protein; (g) {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43For Y43H}, {R44Y or R44L}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G,N49Y, or N49S}, {K53T or K53L}, {L97Y, L97V, or L97I}, {A100I or A100V},and {A107P, A107I, or A107V}; or changes that result in the same aminoacids at the corresponding positions relative to another wild type PD-1protein; and (h) {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43For Y43H}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, or N49S},{K53T or K53L}, {L97Y, L97V, or L97I}, {A100I or A100V}, and {A107P,A107I, or A107V}; or changes that result in the same amino acids at thecorresponding positions relative to another wild type PD-1 protein.

Also provided are high affinity PD-1 mimic polypeptides comprising theamino acid changes, relative to the PD-1 protein fragment set forth inSEQ ID NO: 2, selected from: (a) V39R, N41V, Y43H, M45E, S48G, N49Y,Q50E, K53T, A56S, Q63T, G65L, Q66P, L97V, S102A, L103F, A104H, K106V,and A107I; or changes that result in the same amino acids at thecorresponding positions relative to another wild type PD-1 protein; (b)V39R, N41V, Y43H, M45E, S48N, Q50H, T51A, D52Y, K53T, A56L, Q63L, G65F,Q66P, L97I, S102T, L103F, A104D, K106R, and A107I; or changes thatresult in the same amino acids at the corresponding positions relativeto another wild type PD-1 protein; (c) V39H, L40V, N41V, Y43H, R44Y,M45E, N49G, K53T, M83L, L97V, A100I, and A107I; or changes that resultin the same amino acids at the corresponding positions relative toanother wild type PD-1 protein; (d) V39H, L40V, N41V, Y43H, M45E, N49G,K53T, Q66P, M83L, L97V, and A107I; or changes that result in the sameamino acids at the corresponding positions relative to another wild typePD-1 protein; (e) V39H, L40V, N41V, Y43H, M45E, N49S, K53T, Q66P, H82Q,M83L, L97V, A100V, and A107I; or changes that result in the same aminoacids at the corresponding positions relative to another wild type PD-1protein; (f) V39H, L40I, N41I, Y43H, M45E, N49G, K53T, M83L, L97V,A100V, and A107I; or changes that result in the same amino acids at thecorresponding positions relative to another wild type PD-1 protein; (g)V39H, L40V, N41I, Y43H, R44L, M45E, N49G, K53T, L97V, A100V, and A107I;or changes that result in the same amino acids at the correspondingpositions relative to another wild type PD-1 protein; (h) V39H, L40V,N41I, Y43H, M45E, N49G, K53T, L97V, A100V, and A107I; or changes thatresult in the same amino acids at the corresponding positions relativeto another wild type PD-1 protein; and (i) V39H, L40V, N41V, Y43H, M45E,N49G, K53T, L97V, A100V, and A107I or changes that result in the sameamino acids at the corresponding positions relative to another wild typePD-1 protein.

In some cases, the high affinity PD-1 mimic polypeptide includes (e.g.,is fused to) a fusion partner. In some cases, the fusion partner is afragment of a human immunoglobulin polypeptide sequence (e.g., afragment is selected from: (a) a CH3 domain; and (b) part or whole of anFc region). In some cases the fusion partner is selected from: amultimerization domain; a cytokine; an attenuated cytokine; a 41BB-agonist; CD40-agonist; an inhibitor of BTLA and/or CD160; and aninhibitor of TIM3 and/or CEACAM1.

In some cases, the high affinity PD-1 mimic polypeptide is multimeric(e.g., dimeric). In some such cases, the high affinity PD-1 mimicpolypeptide includes (e.g., is fused to) a fusion partner and the fusionpartner includes a multimerization (e.g., a dimerization) domain. Forexample, in some cases, the fusion partner is a CH3 domain (from aimmunoglobulin polypeptide sequence, e.g., a human immunoglobulinpolypeptide sequence).

In some cases, a subject high affinity PD-1 mimic polypeptide includesone or more mutations corresponding to R87C, N91C, and/or R122C relativeto the PD-1 protein fragment set forth in SEQ ID NO: 2. In some cases, asubject high affinity PD-1 mimic polypeptide includes the amino acidsequence set forth in any of SEQ ID NOs: 3-25 and 39-46. In some cases,the high affinity PD-1 mimic polypeptide includes a detectable label(e.g., a positron-emission tomography (PET) imaging label). In somecases, a subject high affinity PD-1 mimic polypeptide includes one ormore mutations corresponding to R87C, N91C, and/or R122C relative to thePD-1 protein fragment set forth in SEQ ID NO: 2, and also includes adetectable label (e.g., a positron-emission tomography (PET) imaginglabel).

Also provided is a pharmaceutical formulation comprising a subject highaffinity PD-1 mimic polypeptide.

Also provided are nucleic acids. A subject nucleic acid includes anucleotide sequence that encodes a high affinity PD-1 mimic polypeptide.In some cases, the nucleic acid further includes (i) a nucleotidesequence encoding a TCR (e.g., nucleotide sequences encoding a TCR alphapolypeptide and a TCR beta polypeptide of a TCR); and/or (ii) anucleotide sequence encoding a chimeric antigen receptor (CAR). In somecases, the nucleic acid is an expression vector (e.g., a linear vector,a circular vector, a plasmid, a viral vector, etc.).

Also provided are cells that include such a nucleic acid (e.g., humancell, primate cell, mouse cell, mammalian cell)(e.g., an immune cell, aleukocyte, a T cell, a CD8 T cell, a CD4 T cell, a memory/effector Tcell, a B cell, an antigen presenting cell (APC), a dendritic cell, amacrophage, a monocyte, an NK cell, a stem cell, a hematopoietic stemcell, a pluripotent stem cell, a multipotent stem cell, a tissuerestricted stem cell, etc). In some cases, the cell is an immune cell.In some cases, the cell is a stem or progenitor cell. In some cases thecell is a hematopoietic stem cell. In some cases, the cell is a T cell(e.g., a T cell with an engineered T cell receptor (TCR), e.g., achimeric antigen receptor (CAR) T cell).

Also provided are methods of imaging. In some cases, the method includescontacting cells expressing PD-L1 (e.g., in vitro, ex vivo, in vivo)with a subject high affinity PD-1 mimic polypeptide. In some cases, saidcontacting comprises administering the high affinity PD-1 mimicpolypeptide to an individual. In some cases, the method is a method ofdiagnosing and/or prognosing cancer in an individual. Thus, in somecases, the imaging is used for diagnosing and/or prognosing cancer in anindividual.

Also provided are methods of inhibiting the interaction of PD-1 on afirst cell with PD-L1 and/or PD-L2 on a second cell. In some cases, themethod includes contacting the second cell with a high affinity PD-1mimic polypeptide. In some cases, the second cell is a cancer cell or achronically infected cell. In some cases, the contacting is in vitro. Insome cases, the contacting is ex vivo (e.g., one or more cells can beautologous to an individual into whom one or more cells will beintroduced). In some cases, the contacting is in vivo. In some cases,the method includes contacting the second cell with an agent selectedfrom: an immune stimulant, an agent to treat chronic infection, acytotoxic agent, a chemotherapeutic agent, a cell-specific antibody, anantibody selective for a tumor cell marker, and a T cell with anengineered T cell receptor (TCR). In some cases, the method includescontacting the second cell with a tumor specific antibody.

Also provided are methods of treating an individual having cancer,having a chronic infection, or having an immunological disorderassociated with immunosuppression. For example, methods can includeadministering to the individual (e.g., in an amount effective forreducing the binding of PD-1 on a first cell with PD-L1 on a secondcell) a subject high affinity PD-1 mimic polypeptide. In some cases, theadministering includes introducing a nucleic acid encoding the PD-1mimic polypeptide into a third cell. In some cases, the third cell is invivo. In some cases, the nucleic acid encoding the PD-1 mimicpolypeptide is introduced into the third cell in vitro or ex vivo, andthe third cell is then introduced into the individual. In some cases,the third cell is an immune cell. In some cases, the immune cell is a Tcell with an engineered T cell receptor (TCR). In some cases, theengineered TCR is a chimeric antigen receptor (CAR). In some cases, theindividual has an advanced tumor. In some cases, the method includesadministering to the individual an agent selected from: an immunestimulant, an agent to treat chronic infection, a cytotoxic agent, achemotherapeutic agent, a cell-specific antibody, an antibody selectivefor a tumor cell marker, and a T cell with an engineered T cell receptor(TCR).

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIGS. 1A-1B. (FIG. 1A) Schematic illustrating PD-L1 on the surface of atumor cell specifically binding to PD-1 on the surface of a T cell toinhibit activation of the T cell, thereby allowing the tumor cell toevade destruction by the immune system. (FIG. 1B) Schematic illustratinga subject high affinity PD-1 mimic polypeptide specifically binding toPD-L1 on the surface of a cancer cell, thereby reducing the ability ofthe cancer cell to inhibit T cell activation, which in turn reduces thecancer cell's ability to evade the immune response.

FIGS. 2A-2B. (FIG. 2A) Structural representation of the interaction ofPD-1 (upper right) with PD-L1 (lower left). Residues of PD-1 located atthe contact site with PD-L1 are represented as spheres. (FIG. 2B) A PD-1mimic polypeptide (comprising wild type amino acid residues) wasmutagenized at the residues that contact PD-L1 to generate a firstgeneration library (Generation 1), which was displayed on yeast.Selections based on binding were then performed using biotinylated humanPD-L1 (100 nM). A second generation library (Generation 2) that focusedon converging positions was created to screen for PD-1 mimicpolypeptides having even greater affinity for PD-L1 (using 1 nMbiotinylated human PD-L1).

FIG. 3. The table reflects the sequences of the engineered variants(subject high affinity PD-1 mimic polypeptides). “G1” variants are fromGeneration 1 while “G2” variants are from Generation 2 (see FIGS.2A-2B). Each numbered column represents the amino acid position for eachshown residue relative to the PD-1 polypeptide set forth SEQ ID NO: 2(The polypeptide of SEQ ID NO: 2 is a PD-1 mimic polypeptide thatincludes a wild type PD-1 sequence, but lacks a transmembrane domain andlacks the first 25 amino acids of wild type PD-1). Divergence from thewild-type amino acid residue is indicated with the single-letter codefor the resulting mutation for each variant. The measured SurfacePlasmon Resonance (SPR) affinity for PD-L1 is indicated (when measured)at the right. HAC: High Affinity Consensus.

FIG. 4. Two representative Surface Plasmon Resonance (SPR) plots areshown. The dissociation half-life for a native PD-1 mimic polypeptide(having wild-type human PD-1 sequences) was less than one second. Bycontrast, the dissociation half-life for a high-affinity consensus PD-1variant HAC-I (a subject high affinity PD-1 mimic polypeptide) was 42.4minutes.

FIGS. 5A-5C. Subject high affinity PD-1 mimic polypeptides potently andspecifically antagonized PD-L1. Yeast displaying: (FIG. 5A) human PD-L1,(FIG. 5B) human PD-L2, or (FIG. 5C) mouse PD-L1, were stained withlabeled native PD-1 mimic polypeptide streptavidin tetramers (a controlPD-1 mimic polypeptide having wild-type human PD-1 sequences andconjugated to Alexa647). The binding of the labeled native PD-1 mimicpolypeptide to PD-L1 was competed with variable concentrations ofunlabeled high affinity PD-1 mimic polypeptides (concentrationsindicated on the x-axis). (FIG. 5A) An unlabeled native PD-1 mimicpolypeptide (having wild-type human PD-1 sequences) antagonized thePD-1/PD-L1 interaction. High-affinity PD-1 mimic polypeptides (HAC-VPD-1, G2 4-1, and G2 4-2) potently antagonized the PD-1/PD-L1interaction. (FIG. 5B) The high-affinity PD-1 mimic polypeptides did notdemonstrate any antagonism of the PD-1:PD-L2 interaction, while a nativePD-1 mimic polypeptide did antagonize the PD-1:PD-L2 interaction. (FIG.5C) Subject high-affinity PD-1 mimic polypeptides were also able tocompete for binding to mouse PD-L1.

FIGS. 6A-6B. High affinity PD-1 mimic polypeptides antagonize PD-L1 onhuman cancer cells. (FIG. 6A) Expression of PD-L1 on human melanoma cellline SKMEL28. PD-L1 expression was induced on SKMEL28 cells bystimulation with 2000 U/mL human interferon-gamma (IFNγ) for 24 hours.PD-L1 staining was assessed by flow cytometry under induced (plus IFNγ)versus non-induced (minus IFNγ) conditions. (FIG. 6B) IFNγ-stimulatedSKMEL28 cells were stained with labeled native PD-1 mimic polypeptidestreptavidin tetramers (a control PD-1 mimic polypeptide havingwild-type human PD-1 sequences and conjugated to Alexa647) with variableconcentrations of unlabeled high-affinity PD-1 mimic polypeptides(concentrations indicated on the x-axis). An unlabeled native PD-1 mimicpolypeptide (having wild-type human PD-1 sequences) was ineffective atpreventing binding of the labeled native PD-1 mimic polypeptide to theSKMEL28 cells (IC50=8.2 μM). By contrast, HAC-V (a high-affinity PD-1mimic polypeptide) potently inhibited binding of the labeled native PD-1mimic polypeptide (IC50 of 210 pM). HAC-MBH (HAC-V, a high-affinity PD-1mimic polypeptide, fused to the CH3 domain of human IgG1) inhibitedbinding of the labeled native PD-1 mimic polypeptide with additionallyenhanced potency (IC50 of 55 pM).

FIGS. 7A-7C. Directed evolution of high-affinity PD-1 with yeast surfacedisplay. (FIG. 7A) Model of hPD-1 complexed with hPD-L1 constructed bystructural alignment of mPD-1:hPD-L1 complex (PDB ID 3BIK) with hPD-1(PDB ID 3RRQ). Randomized residues of PD-1 are depicted as “bluespheres” for PD-L1 contact residues and “red spheres” for core residues.(FIG. 7B) Histogram overlays assessing yeast hPD-L1 staining at eachround of selection. For the first generation selections (left panel),all rounds were stained with 100 nM biotinylated hPD-L1. For the secondgeneration selections (right panel), yeast were stained with 1 nMbiotinylated hPD-L1. (FIG. 7C) Summary of sequences and hPD-L1affinities for selected PD-1 variants. The position of each mutatedposition and the corresponding residue in wild-type PD-1 is indicated atthe top of the table. Italic font indicates mutations that occurred atnon-randomized sites. Residues indicated as 39, 41, 43, 45, 49, 53, 97,and 107 are PD-L1 contact positions that converged in the HAC consensussequence (“Contact consensus sites”), while residues indicated as 40 and100 are converging core positions (“Core consensus sites”). Theaffinities for some sequences to hPD-L1 were determined (as indicated,far right column) by surface plasmon resonance (SPR).

FIGS. 8A-8B. HAC-PD-1 binds and antagonizes human and murine PD-L1, butnot PD-L2. (FIG. 8A) Representative surface plasmon resonance (SPR)sensorgrams of wild-type PD-1 (left) and HAC-V PD-1 (right) binding toimmobilized hPD-L1. (FIG. 8B) Competition binding assays of wild-typehPD-1, HAC-V PD-1, or HAC ‘microbody’ (HACmb) on human SK-MEL-28 cells(left), mouse B16-F10 cells, or yeast displaying hPD-L2. 100 nMhPD-1/streptavidin-AlexaFluor 647 tetramer was used as the probe ligand.Error bars represent s.e.m.

FIGS. 9A-9D. HAC-PD-1 yields enhanced tumor penetration and does notdeplete peripheral T cells. (FIG. 9A) Representative fluorescencemicroscopy images of sectioned CT26 tumors deficient in PD-L1 (top) ortransgenic for hPD-L1 (bottom) four hours post intraperitoneal injectionof anti-hPD-L1-AlexaFluor488 (green when presented in color) andHAC-AlexaFluor 594 (red when presented in color). Nuclei (blue whenpresented in color) were labeled with DAPI. Scale bars represent 500 μm.(FIG. 9B) Representative flow cytometry of dissociated tumors from FIG.9A showing relative HAC-AlexaFluor 594 staining versusanti-hPD-L1-AlexaFluor 488 staining. Percentages are given in eachpositive quadrant. (FIG. 9C) Summary of flow cytometry studies from 4PD-L1 deficient tumors and 4 hPD-L1 transgenic tumors. n.s., notsignificant. ***, p<0.0001, Two-way ANOVA. Error bars represent s.e.m.(FIG. 9D) Relative abundance of peripheral CD8+ T cells (left-mostpanel), peripheral CD4+ T cells (second from left panel), lymph nodeCD8+ T cells (second from right panel), and lymph node CD4+ T cells(right-most panel) after 3 days of administration of vehicle (PBS),anti-mPD-L1, or HACmb to mice engrafted with CT26 tumors. ns, notsignificant *, p<0.05; ***, p<0.001, One-way ANOVA.

FIGS. 10A-10D. Anti-tumor efficacy of HACmb and anti-PD-L1 antibodies insmall and large CT26 syngeneic tumor models. (FIG. 10A) Schematicillustrating experimental design of small tumor experiment. Treatmentwas initiated for all cohorts 7 days after engraftment of tumors. Micewere injected with daily doses of vehicle (PBS), 250 μg anti-PD-L1(clone 10F.9G2), or 250 μg of HACmb for 14 days. (FIG. 10B) Relativegrowth rates of engrafted tumors, calculated as fold-change fromdisplayed for individual tumors (left three panels) or as summary data(right-most panel) over the course of the treatment period. Error barsrepresent s.e.m. n.s., not significant. ***, p<0.0001. (FIG. 10C)Schematic illustrating experimental design of large tumor experiment.Mice were engrafted with CT26 tumors, and monitored daily. When anindividual tumor exceeded 150 mm³, the mouse was randomized to atreatment cohort. Tumors were measured daily, and received dailytreatment with vehicle (PBS), 250 μg anti-PD-L1 (clone 10F.9G2), or 250μg of HACmb for 14 days. Anti-CTLA4 (clone 9D9) was administered as asingle dose of 250 μg. (FIG. 10D) Summary data for average tumor growthover the 14-day period of treatment. Error bars represent s.e.m. ThePBS-treated tumor growth (black) and anti-CTLA4-treated tumor growth(purple) on the left and right panels are identical; they arerepresented twice for clarity. n.s., not significant. ***, p<0.001,Two-way ANOVA. Complete statistical analysis at day 14 post-treatment isshown in Table 4.

FIGS. 11A-11B. MicroPET imaging of hPD-L1 with ⁶⁴Cu-DOTA-HAC. (FIG. 11A)PET-CT images one hour post injection of ⁶⁴Cu-DOTA-HAC (230 μCi/25μg/200 μl) in NSG mice bearing subcutaneous hPD-L1(+) or hPD-L1(−) CT26tumors. Blocking was performed with 500 μg/200 μl of unlabeled HAC-PD1,2 hours prior to PET tracer. T-tumor, L-liver, K-kidneys, B-bladder,SG-salivary glands. (FIG. 11B) Quantification of tumor uptake one hourpost-injection by region of interest (ROI) analysis, indicated as apercent of injected dose per gram of tissue (% ID/g). *, p<0.05.

FIGS. 12A-12B. Design of the “First Generation” PD-1 library. (FIG. 12A)Table of randomized positions of hPD-1 are given in the table, with thecorresponding degenerate codon and the potential amino acids possible ateach site. (FIG. 12B) Structural depiction of the “First Generation”library; hPD-1 is in green (when presented in color) with randomizedside chains indicated as space-filling spheres.

FIGS. 13A-13B. Design of the “Second Generation” PD-1 library. (FIG.13A) Table of randomized positions of hPD-1 are given in the table, withthe corresponding degenerate codon and the potential amino acidspossible at each site. (FIG. 13B) Structural depiction of the “SecondGeneration” library; hPD-1 is in green (when presented in color) withrandomized side chains indicated as space-filling spheres.

FIG. 14. Schematic diagram of HAC “microbody” (HACmb) design incomparison to individual HAC PD-1 monomer and anti-PD-L1 antibody. HACmbis HAC-V fused to the CH3 domain of human IgG1 linked by adisulfide-containing hinge sequence.

FIGS. 15A-15B. In vitro and in vivo staining of hPD-L1 expressing cells.(FIG. 15A) FACS plot of CT26-Tg(hPD-L1)-Δ(mPDL1) either unstained,stained with AlexaFluor594-labeled HAC monomer, or AlexaFluor488-labeledanti-PD-L1 antibody (clone 29E.2A3, Biolegend). (FIG. 15B) Histologicalsection taken from the same tumor as depicted in FIG. 9A, but from thecenter of the tumor rather than at the periphery. Image is from tumorsdissected four hours after intraperitoneal injection ofanti-hPD-L1-Alexa Fluor488 (green when presented in color) and HAC-AlexaFluor 594 (red when presented in color). Nuclei (blue when presented incolor) were labeled with DAPI. Scale bars represent 500 μm.

FIG. 16. Expression of PD-L1 on primary peripheral blood T cells inCT26-engrafted mice. Dot plot showing the percentage of PD-L1 positiveCD4+ T cells and CD8+ T cells in the peripheral blood of Balb/c hosts 14days post-engraftment with subcutaneous CT26 tumors.

FIGS. 17A-17B. Validation of DOTA-HAC PET tracer. (FIG. 17A) Competitionbinding assays of wild-type hPD-1, HAC-V, or DOTA-HAC on human SK-MEL-28cells. 100 nM hPD-1/streptavidin-Alexa Fluor 647 tetramer was used asthe probe ligand. Error bars represent s.e.m. (FIG. 17B)Immunoreactivity of anti-hPD-L1 radiotracer. hPD-L1(+), hPD-L1(−) andhPD-L1(+) cells blocked with excess HAC-N91C prior to the addition oftracer were tested for binding specificity. 5 nM ⁶⁴Cu-DOTA-HAC readilybound to hPD-L1(+) cells (80.5%±1.9%), while control hPD-L1(−) cellsonly exhibited minimal immunoreactivity (8.3%±0.5%). Binding was blockedin hPD-L1(+) cells by the addition of HAC-N91C to 1 μM (8.9%±0.1%).n.s., not significant. ****, p<0.0001, Two-way ANOVA.

FIGS. 18A-18E. ⁶⁴Cu-DOTA-HAC MicroPET imaging dynamics. (FIG. 18A) Tumoruptake computed by region of interest (ROI) analysis over 24 hours.(FIG. 18B) Renal uptake in hPD-L1 (+) and (−) tumor bearing miceassessed by ROI analysis. (FIG. 18C) PET-CT image 24 hourspost-injection of ⁶⁴Cu-DOTA-HAC (230 μCi/25 μg/200 μl) in NSG mousebearing dual subcutaneous hPD-L1(+) (dashed) and hPD-L1(−) (solid) CT26tumors. (FIG. 18D) Tumor uptake computed by region of interest (ROI).(FIG. 18E) Renal clearance over 24 h. Uptake values given as percentageof injected dose per gram of tissue (% ID/g).

FIGS. 19A-19B. 24 hour biodistribution of ⁶⁴Cu-DOTA-HAC. (FIG. 19A)After completion of micro-PET/CT imaging, mice were euthanized anddissected for biodistribution. Uptake in the indicated organs andtissues are given as the percentage of injected dose per gram of tissue(% ID/g). (FIG. 19B) Relative amount of hPD-L1(+) tumor radiotraceruptake compared to blood and muscle.

FIGS. 20A-20E. Schematic depiction of examples of viral vectorconstructs encoding a subject high affinity PD-1 mimic polypeptide aswell as (i) a heterologous TCR (that binds to an antigen)(e.g., encodesthe TCR-alpha and TCR-beta polypeptides) or (ii) a chimeric antigenreceptor (CAR). FIG. 20A provides a legend for FIGS. 20B-20E.

FIGS. 21A-21D. Schematic depiction of example nucleic acids (DNAvectors) encoding a subject high affinity PD-1 mimic polypeptide.

DETAILED DESCRIPTION

High affinity PD-1 mimic polypeptides, and methods of their use, areprovided. The high affinity PD-1 mimic polypeptides are sequencevariants of a wild type PD-1 protein (e.g., the wild type human PD-1protein), and have utility for in vivo and in vitro methods that blockthe interaction between a wild type PD-1 protein and its ligand PD-L(PD-L1 and/or PD-L2). A high affinity PD-1 mimic polypeptide includes atleast one amino acid change relative to a wild-type PD-1 protein, has anincreased affinity for PD-L (PD-L1 and/or PD-L2) relative to thewild-type PD-1 protein, and lacks a transmembrane domain of a wild typePD-1 protein. The amino acid changes that provide for increased affinitycan be localized to amino acid positions of contact between PD-1 andPD-L, and/or can be located in the immunoglobulin domain of PD-1 proteinfrom which it was derived.

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to particular method orcomposition described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof,e.g., polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DEFINITIONS

In the description that follows, a number of terms conventionally usedin the field are utilized. In order to provide a clear and consistentunderstanding of the specification and claims, and the scope to be givento such terms, the following definitions are provided.

The terms “inhibitors,” “blocking agents” and “masking agents” of theinteraction between PD-1 and its ligand PD-L1 refer to molecules thatprevent the binding of PD-1 and PD-L1. For development purposes thebinding may be performed under experimental conditions, e.g., usingisolated proteins as binding partners, using portions of proteins asbinding partners, using yeast display of proteins or portions ofproteins as binding partners, and the like.

For physiologically relevant purposes the binding of PD-1 and PD-L1 isusually an event between two cells, where each cell expresses one of thebinding partners. In some cases, PD-1 is expressed on the surface ofimmune cells (e.g., T cells), and PD-L1 is expressed on cells that couldbe targets for destruction by the immune system (e.g., tumor cells,cells harboring an infection such as a chronic infection, and the like).Inhibitors may be identified using in vitro and in vivo assays forreceptor or ligand binding or signaling.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms also apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an .alpha. carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

The terms “recipient”, “individual”, “subject”, “host”, and “patient”,are used interchangeably herein and refer to any mammalian subject forwhom diagnosis, treatment, or therapy is desired, particularly humans.“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc.In some embodiments, the mammal is human.

The terms “cancer,” “neoplasm,” and “tumor” are used interchangeablyherein to refer to cells which exhibit autonomous, unregulated growth,such that they exhibit an aberrant growth phenotype characterized by asignificant loss of control over cell proliferation. Cells of interestfor detection, analysis, or treatment in the present application includeprecancerous (e.g., benign), malignant, pre-metastatic, metastatic, andnon-metastatic cells. Cancers of virtually every tissue are known. Thephrase “cancer burden” refers to the quantum of cancer cells or cancervolume in a subject. Reducing cancer burden accordingly refers toreducing the number of cancer cells or the cancer volume in a subject.The term “cancer cell” as used herein refers to any cell that is acancer cell or is derived from a cancer cell e.g., clone of a cancercell. Many types of cancers are known to those of skill in the art,including solid tumors such as carcinomas, sarcomas, glioblastomas,melanomas, lymphomas, myelomas, etc., and circulating cancers such asleukemias.

As used herein “cancer” includes any form of cancer, including but notlimited to solid tumor cancers (e.g., lung, prostate, breast, bladder,colon, ovarian, pancreas, kidney, liver, glioblastoma, medulloblastoma,leiomyosarcoma, head & neck squamous cell carcinomas, melanomas,neuroendocrine; etc.) and liquid cancers (e.g., hematological cancers);carcinomas; soft tissue tumors; sarcomas; teratomas; melanomas;leukemias; lymphomas; and brain cancers, including minimal residualdisease, and including both primary and metastatic tumors. Any cancer isa suitable cancer to be treated by the subject methods and compositions.In some cases, the cancer cells express PD-L1. In some cases, the cancercells do not express PD-L1 (e.g., in such cases, cells of the immunesystem of the individual being treated express PD-L1).

Carcinomas are malignancies that originate in the epithelial tissues.Epithelial cells cover the external surface of the body, line theinternal cavities, and form the lining of glandular tissues. Examples ofcarcinomas include, but are not limited to: adenocarcinoma (cancer thatbegins in glandular (secretory) cells), e.g., cancers of the breast,pancreas, lung, prostate, and colon can be adenocarcinomas;adrenocortical carcinoma; hepatocellular carcinoma; renal cellcarcinoma; ovarian carcinoma; carcinoma in situ; ductal carcinoma;carcinoma of the breast; basal cell carcinoma; squamous cell carcinoma;transitional cell carcinoma; colon carcinoma; nasopharyngeal carcinoma;multilocular cystic renal cell carcinoma; oat cell carcinoma; large celllung carcinoma; small cell lung carcinoma; non-small cell lungcarcinoma; and the like. Carcinomas may be found in prostrate, pancreas,colon, brain (usually as secondary metastases), lung, breast, skin, etc.

Soft tissue tumors are a highly diverse group of rare tumors that arederived from connective tissue. Examples of soft tissue tumors include,but are not limited to: alveolar soft part sarcoma; angiomatoid fibroushistiocytoma; chondromyoxid fibroma; skeletal chondrosarcoma;extraskeletal myxoid chondrosarcoma; clear cell sarcoma; desmoplasticsmall round-cell tumor; dermatofibrosarcoma protuberans; endometrialstromal tumor; Ewing's sarcoma; fibromatosis (Desmoid); fibrosarcoma,infantile; gastrointestinal stromal tumor; bone giant cell tumor;tenosynovial giant cell tumor; inflammatory myofibroblastic tumor;uterine leiomyoma; leiomyosarcoma; lipoblastoma; typical lipoma; spindlecell or pleomorphic lipoma; atypical lipoma; chondroid lipoma;well-differentiated liposarcoma; myxoid/round cell liposarcoma;pleomorphic liposarcoma; myxoid malignant fibrous histiocytoma;high-grade malignant fibrous histiocytoma; myxofibrosarcoma; malignantperipheral nerve sheath tumor; mesothelioma; neuroblastoma;osteochondroma; osteosarcoma; primitive neuroectodermal tumor; alveolarrhabdomyosarcoma; embryonal rhabdomyosarcoma; benign or malignantschwannoma; synovial sarcoma; Evan's tumor; nodular fasciitis;desmoid-type fibromatosis; solitary fibrous tumor; dermatofibrosarcomaprotuberans (DFSP); angiosarcoma; epithelioid hemangioendothelioma;tenosynovial giant cell tumor (TGCT); pigmented villonodular synovitis(PVNS); fibrous dysplasia; myxofibrosarcoma; fibrosarcoma; synovialsarcoma; malignant peripheral nerve sheath tumor; neurofibroma; andpleomorphic adenoma of soft tissue; and neoplasias derived fromfibroblasts, myofibroblasts, histiocytes, vascular cells/endothelialcells and nerve sheath cells.

A sarcoma is a rare type of cancer that arises in cells of mesenchymalorigin, e.g., in bone or in the soft tissues of the body, includingcartilage, fat, muscle, blood vessels, fibrous tissue, or otherconnective or supportive tissue. Different types of sarcoma are based onwhere the cancer forms. For example, osteosarcoma forms in bone,liposarcoma forms in fat, and rhabdomyosarcoma forms in muscle. Examplesof sarcomas include, but are not limited to: askin's tumor; sarcomabotryoides; chondrosarcoma; ewing's sarcoma; malignanthemangioendothelioma; malignant schwannoma; osteosarcoma; and softtissue sarcomas (e.g., alveolar soft part sarcoma; angiosarcoma;cystosarcoma phyllodesdermatofibrosarcoma protuberans (DFSP); desmoidtumor; desmoplastic small round cell tumor; epithelioid sarcoma;extraskeletal chondrosarcoma; extraskeletal osteosarcoma; fibrosarcoma;gastrointestinal stromal tumor (GIST); hemangiopericytoma;hemangiosarcoma (more commonly referred to as “angiosarcoma”); kaposi'ssarcoma; leiomyosarcoma; liposarcoma; lymphangiosarcoma; malignantperipheral nerve sheath tumor (MPNST); neurofibrosarcoma; synovialsarcoma; undifferentiated pleomorphic sarcoma, and the like).

A teratoma is a type of germ cell tumor that may contain severaldifferent types of tissue (e.g., can include tissues derived from anyand/or all of the three germ layers: endoderm, mesoderm, and ectoderm),including for example, hair, muscle, and bone. Teratomas occur mostoften in the ovaries in women, the testicles in men, and the tailbone inchildren.

Melanoma is a form of cancer that begins in melanocytes (cells that makethe pigment melanin). It may begin in a mole (skin melanoma), but canalso begin in other pigmented tissues, such as in the eye or in theintestines.

Leukemias are cancers that start in blood-forming tissue, such as thebone marrow, and causes large numbers of abnormal blood cells to beproduced and enter the bloodstream. For example, leukemias can originatein bone marrow-derived cells that normally mature in the bloodstream.Leukemias are named for how quickly the disease develops and progresses(e.g., acute versus chronic) and for the type of white blood cell thatis affected (e.g., myeloid versus lymphoid). Myeloid leukemias are alsocalled myelogenous or myeloblastic leukemias. Lymphoid leukemias arealso called lymphoblastic or lymphocytic leukemia. Lymphoid leukemiacells may collect in the lymph nodes, which can become swollen. Examplesof leukemias include, but are not limited to: Acute myeloid leukemia(AML), Acute lymphoblastic leukemia (ALL), Chronic myeloid leukemia(CML), and Chronic lymphocytic leukemia (CLL).

Lymphomas are cancers that begin in cells of the immune system. Forexample, lymphomas can originate in bone marrow-derived cells thatnormally mature in the lymphatic system. There are two basic categoriesof lymphomas. One kind is Hodgkin lymphoma (HL), which is marked by thepresence of a type of cell called the Reed-Sternberg cell. There arecurrently 6 recognized types of HL. Examples of Hodgkin lymphomasinclude: nodular sclerosis classical Hodgkin lymphoma (CHL), mixedcellularity CHL, lymphocyte-depletion CHL, lymphocyte-rich CHL, andnodular lymphocyte predominant HL.

The other category of lymphoma is non-Hodgkin lymphomas (NHL), whichincludes a large, diverse group of cancers of immune system cells.Non-Hodgkin lymphomas can be further divided into cancers that have anindolent (slow-growing) course and those that have an aggressive(fast-growing) course. There are currently 61 recognized types of NHL.Examples of non-Hodgkin lymphomas include, but are not limited to:AIDS-related Lymphomas, anaplastic large-cell lymphoma,angioimmunoblastic lymphoma, blastic NK-cell lymphoma, Burkitt'slymphoma, Burkitt-like lymphoma (small non-cleaved cell lymphoma),chronic lymphocytic leukemia/small lymphocytic lymphoma, cutaneousT-Cell lymphoma, diffuse large B-Cell lymphoma, enteropathy-type T-Celllymphoma, follicular lymphoma, hepatosplenic gamma-delta T-Celllymphomas, T-Cell leukemias, lymphoblastic lymphoma, mantle celllymphoma, marginal zone lymphoma, nasal T-Cell lymphoma, pediatriclymphoma, peripheral T-Cell lymphomas, primary central nervous systemlymphoma, transformed lymphomas, treatment-related T-Cell lymphomas, andWaldenstrom's macroglobulinemia.

Brain cancers include any cancer of the brain tissues. Examples of braincancers include, but are not limited to: gliomas (e.g., glioblastomas,astrocytomas, oligodendrogliomas, ependymomas, and the like),meningiomas, pituitary adenomas, vestibular schwannomas, primitiveneuroectodermal tumors (medulloblastomas), etc.

The “pathology” of cancer includes all phenomena that compromise thewell-being of the patient. This includes, without limitation, abnormalor uncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, neoplasia, premalignancy,malignancy, invasion of surrounding or distant tissues or organs, suchas lymph nodes, etc.

As used herein, the terms “cancer recurrence” and “tumor recurrence,”and grammatical variants thereof, refer to further growth of neoplasticor cancerous cells after diagnosis of cancer. Particularly, recurrencemay occur when further cancerous cell growth occurs in the canceroustissue. “Tumor spread,” similarly, occurs when the cells of a tumordisseminate into local or distant tissues and organs; therefore tumorspread encompasses tumor metastasis. “Tumor invasion” occurs when thetumor growth spread out locally to compromise the function of involvedtissues by compression, destruction, or prevention of normal organfunction.

As used herein, the term “metastasis” refers to the growth of acancerous tumor in an organ or body part, which is not directlyconnected to the organ of the original cancerous tumor. Metastasis willbe understood to include micrometastasis, which is the presence of anundetectable amount of cancerous cells in an organ or body part which isnot directly connected to the organ of the original cancerous tumor.Metastasis can also be defined as several steps of a process, such asthe departure of cancer cells from an original tumor site, and migrationand/or invasion of cancer cells to other parts of the body.

The term “sample” with respect to a patient encompasses blood and otherliquid samples of biological origin, solid tissue samples such as abiopsy specimen or tissue cultures or cells derived therefrom and theprogeny thereof. The definition also includes samples that have beenmanipulated in any way after their procurement, such as by treatmentwith reagents; washed; or enrichment for certain cell populations, suchas cancer cells. The definition also includes sample that have beenenriched for particular types of molecules, e.g., nucleic acids,polypeptides, etc. The term “biological sample” encompasses a clinicalsample, and also includes tissue obtained by surgical resection, tissueobtained by biopsy, cells in culture, cell supernatants, cell lysates,tissue samples, organs, bone marrow, blood, plasma, serum, and the like.A “biological sample” includes a sample obtained from a patient's cancercell, e.g., a sample comprising polynucleotides and/or polypeptides thatis obtained from a patient's cancer cell (e.g., a cell lysate or othercell extract comprising polynucleotides and/or polypeptides); and asample comprising cancer cells from a patient. A biological samplecomprising a cancer cell from a patient can also include non-cancerouscells.

The term “diagnosis” is used herein to refer to the identification of amolecular or pathological state, disease or condition, such as theidentification of a molecular subtype of breast cancer, prostate cancer,or other type of cancer.

The term “prognosis” is used herein to refer to the prediction of thelikelihood of disease progression (e.g., cancer-attributable death orprogression), including recurrence, metastatic spread of cancer, anddrug resistance. The term “prediction” is used herein to refer to theact of foretelling or estimating, based on observation, experience, orscientific reasoning. In one example, a physician may predict thelikelihood that a patient will survive, following surgical removal of aprimary tumor and/or chemotherapy for a certain period of time withoutcancer recurrence.

The terms “specific binding,” “specifically binds,” and the like, referto non-covalent or covalent preferential binding to a molecule relativeto other molecules or moieties in a solution or reaction mixture (e.g.,an antibody specifically binds to a particular polypeptide or epitoperelative to other available polypeptides). In some embodiments, theaffinity of one molecule for another molecule to which it specificallybinds is characterized by a K_(d) (dissociation constant) of 10⁻⁵ M orless (e.g., 10⁻⁶ M or less, 10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ M orless, 10⁻¹⁰ M or less, 10⁻¹¹ M or less, 10⁻¹² M or less, 10⁻¹³ M orless, 10⁻¹⁴ M or less, 10⁻¹⁵ M or less, or 10⁻¹⁶ M or less). “Affinity”refers to the strength of binding, increased binding affinity beingcorrelated with a lower K_(d).

The term “specific binding member” as used herein refers to a member ofa specific binding pair (i.e., two molecules, usually two differentmolecules, where one of the molecules, e.g., a first specific bindingmember, through non-covalent means specifically binds to the othermolecule, e.g., a second specific binding member).

The terms “co-administration”, “co-administer”, and “in combinationwith” include the administration of two or more therapeutic agentseither simultaneously, concurrently or sequentially within no specifictime limits. In one embodiment, the agents are present in the cell or inthe subject's body at the same time or exert their biological ortherapeutic effect at the same time. In one embodiment, the therapeuticagents are in the same composition or unit dosage form. In otherembodiments, the therapeutic agents are in separate compositions or unitdosage forms. In certain embodiments, a first agent can be administeredprior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes,15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) theadministration of a second therapeutic agent.

For example, “Concomitant administration” of a cancer therapeutic drug,therapeutic drug to treat an infection, or tumor-directed antibody, witha pharmaceutical composition of the present disclosure meansadministration with the high affinity PD-1 mimic polypeptide at suchtime that both the drug/antibody and the composition of the presentdisclosure will have a therapeutic effect. Such concomitantadministration may involve concurrent (i.e. at the same time), prior, orsubsequent administration of the drug/antibody with respect to theadministration of a compound of the disclosure. A person of ordinaryskill in the art would have no difficulty determining the appropriatetiming, sequence and dosages of administration for particular drugs andcompositions of the present disclosure.

In some embodiments, treatment is accomplished by administering acombination of a high affinity PD-1 mimic polypeptide of the disclosurewith another agent (e.g., an immune stimulant, an agent to treat chronicinfection, a cytotoxic agent, etc.). One exemplary class of cytotoxicagents are chemotherapeutic agents. Exemplary chemotherapeutic agentsinclude, but are not limited to, aldesleukin, altretamine, amifostine,asparaginase, bleomycin, capecitabine, carboplatin, carmustine,cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine,dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol,duocarmycin, etoposide, filgrastim, fludarabine, fluorouracil,gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide,interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin,megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane,mitoxantrone, omeprazole, ondansetron, paclitaxel (Taxol™), pilocarpine,prochloroperazine, rituximab, saproin, tamoxifen, taxol, topotecanhydrochloride, trastuzumab, vinblastine, vincristine and vinorelbinetartrate.

Other combination therapies include administration with cell-specificantibodies, for example antibodies selective for tumor cell markers,radiation, surgery, and/or hormone deprivation (Kwon et al., Proc. Natl.Acad. Sci U.S.A., 96: 15074-9, 1999). Angiogenesis inhibitors can alsobe combined with the methods of the disclosure. A number of antibodiesare currently in clinical use for the treatment of cancer, and othersare in varying stages of clinical development. For example, there are anumber of antigens and corresponding monoclonal antibodies for thetreatment of B cell malignancies. One target antigen is CD20. Rituximabis a chimeric unconjugated monoclonal antibody directed at the CD20antigen. CD20 has an important functional role in B cell activation,proliferation, and differentiation. The CD52 antigen is targeted by themonoclonal antibody alemtuzumab, which is indicated for treatment ofchronic lymphocytic leukemia. CD22 is targeted by a number ofantibodies, and has recently demonstrated efficacy combined with toxinin chemotherapy-resistant hairy cell leukemia. Two new monoclonalantibodies targeting CD20, tositumomab and ibritumomab, have beensubmitted to the Food and Drug Administration (FDA). These antibodiesare conjugated with radioisotopes. Alemtuzumab (Campath) is used in thetreatment of chronic lymphocytic leukemia; Gemtuzumab (Mylotarg) findsuse in the treatment of acute myelogenous leukemia; Ibritumomab(Zevalin) finds use in the treatment of non-Hodgkin's lymphoma;Panitumumab (Vectibix) finds use in the treatment of colon cancer.

Monoclonal antibodies, including humanized and chimeric variants, usefulin the methods of the disclosure that have been used in solid tumorsinclude, without limitation, edrecolomab and trastuzumab (herceptin).Edrecolomab targets the 17-1A antigen seen in colon and rectal cancer,and has been approved for use in Europe for these indications.Trastuzumab targets the HER-2/neu antigen. This antigen is seen on 25%to 35% of breast cancers. Cetuximab (Erbitux) is also of interest foruse in the methods of the disclosure. The antibody binds to the EGFreceptor (EGFR), and has been used in the treatment of solid tumorsincluding colon cancer and squamous cell carcinoma of the head and neck(SCCHN).

As such, in some cases, a subject high affinity PD-1 mimic polypeptideis co-administered with an agent (e.g., an antibody) that specificallybinds an antigen other than PD-L1 (e.g., CD19, CD20, CD22, CD24, CD25,CD30, CD33, CD38, CD44, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279(PD-1), EGFR, HER2, CD117, C-Met, PTHR2, HAVCR2 (TIM3), etc.) Examplesof antibodies with CDRs that provide specific binding to a cancer cellmarker (and therefore can be used in a combination therapy(co-administered with a subject high affinity PD-1 mimic polypeptide)include, but are not limited to: CETUXIMAB (binds EGFR), PANITUMUMAB(binds EGFR), RITUXIMAB (binds CD20), TRASTUZUMAB (binds HER2),PERTUZUMAB (binds HER2), ALEMTUZUMAB (binds CD52), and BRENTUXIMAB(binds CD30).

In some cases, a subject high affinity PD-1 mimic polypeptide isco-administered with a T cell with an engineered T cell receptor (TCR)(such a cell is also referred to herein as a “TCR-engineered T cell”).Non-limiting suitable examples of a TCR-engineered T cell are: (i) a Tcell that includes a chimeric antigen receptor (CAR); and (ii) a T cellthat includes a heterologous TCR that binds to an antigen such as acancer antigen. (TCR-engineered T cells are described in more detailbelow in the section on introducing nucleic acids).

A subject high affinity PD-1 mimic polypeptide can be co-administeredwith any convenient immunomodulatory agent (e.g., an anti-CTLA4antibody, an anti-PD-1 antibody, a CD40 agonist, a 4-1 BB modulator(e.g., a 41 BB-agonist), and the like). In some cases, a subject highaffinity PD-1 mimic polypeptide is co-administered with an inhibitor ofBTLA and/or CD160. In some cases, a subject high affinity PD-1 mimicpolypeptide is co-administered with an inhibitor of TIM3 and/or CEACAM1.

As used herein, the phrase “disease-free survival,” refers to the lackof such tumor recurrence and/or spread and the fate of a patient afterdiagnosis, with respect to the effects of the cancer on the life-span ofthe patient. The phrase “overall survival” refers to the fate of thepatient after diagnosis, despite the possibility that the cause of deathin a patient is not directly due to the effects of the cancer. Thephrases, “likelihood of disease-free survival”, “risk of recurrence” andvariants thereof, refer to the probability of tumor recurrence or spreadin a patient subsequent to diagnosis of cancer, wherein the probabilityis determined according to the process of the disclosure.

As used herein, the term “correlates,” or “correlates with,” and liketerms, refers to a statistical association between instances of twoevents, where events include numbers, data sets, and the like. Forexample, when the events involve numbers, a positive correlation (alsoreferred to herein as a “direct correlation”) means that as oneincreases, the other increases as well. A negative correlation (alsoreferred to herein as an “inverse correlation”) means that as oneincreases, the other decreases.

“Dosage unit” refers to physically discrete units suited as unitarydosages for the particular individual to be treated. Each unit cancontain a predetermined quantity of active compound(s) calculated toproduce the desired therapeutic effect(s) in association with therequired pharmaceutical carrier. The specification for the dosage unitforms can be dictated by (a) the unique characteristics of the activecompound(s) and the particular therapeutic effect(s) to be achieved, and(b) the limitations inherent in the art of compounding such activecompound(s).

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous. The terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a human without the production of undesirablephysiological effects to a degree that would prohibit administration ofthe composition.

A “therapeutically effective amount” means the amount that, whenadministered to a subject for treating a disease, is sufficient toeffect treatment for that disease.

The term “target cell” as used herein refers to a cell targeted fordestruction by the immune system after administration of a subject highaffinity PD-1 mimic polypeptide. In some cases, the target cellexpresses a PD-L protein (e.g., PD-L1 and/or PD-L2). A subject highaffinity PD-1 mimic polypeptide can bind to a target cell by virtue ofbinding to the PD-L protein expressed on the surface of the target cell.Thus, the term “target cell” can refer to a PD-L1-expressing cellbecause a subject high affinity PD-1 mimic polypeptide, by inhibitingthe interaction between the PD-L1 expressing cell and the PD-1expressing cell, facilitates decreased PD-1 signaling in the PD-1expressing cell.

However, a target cell need not express PD-L1. In some cases a targetcell (e.g., an infected cell, a cancer cell, etc.) does not expressPD-L1. In some such cases, administration of a subject high affinityPD-1 mimic polypeptide leads to stimulation of the immune system,thereby leading to the destruction of the target cell.

In some cases, a target cell is an “inflicted” cell (e.g., a cell froman “inflicted” individual), where the term “inflicted” is used herein torefer to a subject with symptoms, an illness, or a disease that can betreated with a subject high affinity PD-1 mimic polypeptide. An“inflicted” individual can have cancer, can harbor an infection (e.g., achronic infection), can have an immunological disorder (e.g., a disorderassociated with immunosuppression), can have an inflammatory disorder,and/or can have other hyper-proliferative conditions, for examplesclerosis, fibrosis, and the like, etc. “Inflicted cells” can be thosecells that cause the symptoms, illness, or disease. As non-limitingexamples, the inflicted cells of an inflicted patient can be PD-L1expressing cancer cells, infected cells, inflammatory cells, and thelike. In some cases, one indication that an illness or disease can betreated with a subject high affinity PD-1 mimic polypeptide is that theinvolved cells (i.e., the inflicted cells, e.g., the cancerous cells,the infected cells, the inflammatory cells, the immune cells, etc.)express PD-L1. In some cases, the inflicted cell (e.g., cancer cells) donot express PD-L1, but the disease (e.g., cancer) can still be treatedusing a subject high affinity PD-1 mimic polypeptide.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect can be prophylactic in terms ofcompletely or partially preventing a disease or symptom(s) thereofand/or may be therapeutic in terms of a partial or completestabilization or cure for a disease and/or adverse effect attributableto the disease. The term “treatment” encompasses any treatment of adisease in a mammal, particularly a human, and includes: (a) preventingthe disease and/or symptom(s) from occurring in a subject who may bepredisposed to the disease or symptom but has not yet been diagnosed ashaving it; (b) inhibiting the disease and/or symptom(s), i.e., arrestingtheir development; or (c) relieving the disease symptom(s), i.e.,causing regression of the disease and/or symptom(s). Those in need oftreatment include those already inflicted (e.g., those with cancer,those with an infection, those with an immune disorder, etc.) as well asthose in which prevention is desired (e.g., those with increasedsusceptibility to cancer, those with an increased likelihood ofinfection, those suspected of having cancer, those suspected ofharboring an infection, etc.).

A therapeutic treatment is one in which the subject is inflicted priorto administration and a prophylactic treatment is one in which thesubject is not inflicted prior to administration. In some embodiments,the subject has an increased likelihood of becoming inflicted or issuspected of being inflicted prior to treatment. In some embodiments,the subject is suspected of having an increased likelihood of becominginflicted.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to a subject PD-1mimic polypeptide. The label may itself be detectable by itself (e.g.,radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

By “solid phase” is meant a non-aqueous matrix to which a high affinityPD-1 mimic polypeptide of the present disclosure can adhere. Examples ofsolid phases encompassed herein include those formed partially orentirely of glass (e.g., controlled pore glass), polysaccharides (e.g.,agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.

In certain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity. “Antibodies” (Abs) and“immunoglobulins” (Igs) are glycoproteins having the same structuralcharacteristics. While antibodies exhibit binding specificity to aspecific antigen, immunoglobulins include both antibodies and otherantibody-like molecules which lack antigen specificity. Polypeptides ofthe latter kind are, for example, produced at low levels by the lymphsystem and at increased levels by myelomas.

“Antibody fragment”, and all grammatical variants thereof, as usedherein are defined as a portion of an intact antibody comprising theantigen binding site or variable region of the intact antibody, whereinthe portion is free of the constant heavy chain domains (i.e. CH2, CH3,and CH4, depending on antibody isotype) of the Fc region of the intactantibody. Examples of antibody fragments include Fab, Fab′, Fab′-SH,F(ab′)₂, and Fv fragments; diabodies; any antibody fragment that is apolypeptide having a primary structure consisting of one uninterruptedsequence of contiguous amino acid residues (referred to herein as a“single-chain antibody fragment” or “single chain polypeptide”),including without limitation (1) single-chain Fv (scFv) molecules (2)single chain polypeptides containing only one light chain variabledomain, or a fragment thereof that contains the three CDRs of the lightchain variable domain, without an associated heavy chain moiety (3)single chain polypeptides containing only one heavy chain variableregion, or a fragment thereof containing the three CDRs of the heavychain variable region, without an associated light chain moiety and (4)nanobodies comprising single Ig domains from non-human species or otherspecific single-domain binding modules; and multispecific or multivalentstructures formed from antibody fragments. In an antibody fragmentcomprising one or more heavy chains, the heavy chain(s) can contain anyconstant domain sequence (e.g., CH1 in the IgG isotype) found in anon-Fc region of an intact antibody, and/or can contain any hinge regionsequence found in an intact antibody, and/or can contain a leucinezipper sequence fused to or situated in the hinge region sequence or theconstant domain sequence of the heavy chain(s).

“Native antibodies and immunoglobulins” are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (V_(H)) followed by a number of constant domains. Eachlight chain has a variable domain at one end (V_(L)) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and the lightchain variable domain is aligned with the variable domain of the heavychain. Particular amino acid residues are believed to form an interfacebetween the light- and heavy-chain variable domains (Clothia et al., J.Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci.U.S.A. 82:4592 (1985)).

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a b-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the b-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, National Institute ofHealth, Bethesda, Md. (1991)). The constant domains are not involveddirectly in binding an antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species(scFv), one heavy- and one light-chain variable domain can be covalentlylinked by a flexible peptide linker such that the light and heavy chainscan associate in a “dimeric” structure analogous to that in a two-chainFv species. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, IgA₂. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called a, d, e, g, and m, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known. Engineered variants of immunoglobulinsubclasses, including those that increase or decrease immune effectorfunctions, half-life, or serum-stability, are also encompassed by thisterminology.

Unless specifically indicated to the contrary, the term “conjugate” asdescribed and claimed herein is defined as a heterogeneous moleculeformed by the covalent attachment of one or more antibody fragment(s) toone or more polymer molecule(s), wherein the heterogeneous molecule iswater soluble, i.e. soluble in physiological fluids such as blood, andwherein the heterogeneous molecule is free of any structured aggregate.A conjugate of interest is PEG. In the context of the foregoingdefinition, the term “structured aggregate” refers to (1) any aggregateof molecules in aqueous solution having a spheroid or spheroid shellstructure, such that the heterogeneous molecule is not in a micelle orother emulsion structure, and is not anchored to a lipid bilayer,vesicle or liposome; and (2) any aggregate of molecules in solid orinsolubilized form, such as a chromatography bead matrix, that does notrelease the heterogeneous molecule into solution upon contact with anaqueous phase. Accordingly, the term “conjugate” as defined hereinencompasses the aforementioned heterogeneous molecule in a precipitate,sediment, bioerodible matrix or other solid capable of releasing theheterogeneous molecule into aqueous solution upon hydration of thesolid.

As used in this disclosure, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

Compositions

High affinity PD-1 mimic polypeptides and analogs thereof are provided,which may be referred to generically as high affinity PD-1 reagents. Thehigh affinity PD-1 mimic polypeptides are variants of the wild typehuman PD-1 protein. In some embodiments, the present disclosure providesa high affinity PD-1 mimic polypeptide, where the polypeptide lacks thePD-1 transmembrane domain (and can be a soluble high affinity PD-1 mimicpolypeptide) and includes at least one amino acid change relative to thewild-type PD-1 sequence, and where the amino acid change increases theaffinity of the PD-1 mimic polypeptide for binding to PD-L1 (e.g., bydecreasing the off-rate by at least 10-fold, at least 20-fold, at least50-fold, at least 100-fold, at least 500-fold, or more).

It is to be understood that when a subject high affinity PD-1 mimicpolypeptide lacks a PD-1 transmembrane domain, some amino acids from atransmembrane domain (e.g., a PD-1 transmembrane domain) may still bepresent (e.g., some amino acids from the transmembrane may be retained,as long as the protein retains the desired function). In some cases, asubject high affinity PD-1 mimic polypeptide is soluble. In some cases,a subject high affinity PD-1 mimic polypeptide lacks a PD-1transmembrane domain, but includes a heterologous transmembrane domain(i.e., a transmembrane domain form a protein other than PD-1). In somecases, a subject high affinity PD-1 mimic polypeptide includes atransmembrane domain (e.g., a heterologous transmembrane domain, a PD-1transmembrane domain, etc.), and includes a cleavable linker between theectodomain portion and the transmembrane domain.

Polypeptides

A “PD-1 mimic polypeptide” as used herein refers to a polypeptide havingthe portion of a PD-1 protein that is sufficient to bind PD-L (e.g.,PD-L1 and/or PD-L2) at a recognizable affinity, but which lacks atransmembrane domain (e.g., lacks the naturally present transmembranedomain of a wild type PD-1 protein). Thus, unlike a naturally existingPD-1 polypeptide, a subject PD-1 mimic polypeptide is not permanentlytethered to a cell membrane by way of a transmembrane domain. In someembodiments, a subject PD-1 mimic polypeptide is soluble. Anextracellular domain of protein that is normally tethered to the plasmamembrane of a cell is sometimes referred to in the art as an ectodomain.Thus, a PD-1 mimic polypeptide can be considered to be (or be derivedfrom) an ectodomain of PD-1, or can be considered to include at least aportion of (or a portion that is derived from) the ectodomain of a PD-1polypeptide.

A wild type PD-1 protein has a transmembrane domain, is expressed on thecell surface, and specifically binds to its PD-L ligands (PD-L1 andPD-L2), which are also expressed on the cell surface. Thus, a wild typePD-1, expressed on the surface of a first cell, specifically binds toPD-L1 and/or PD-L2, expressed on the surface of a second cell. A PD-1protein, to which a PD-1 mimic polypeptide corresponds (e.g., from whicha PD-1 mimic polypeptide is derived) can be any PD-1 protein (e.g., awild type PD-1 protein). Example PD-1 proteins include those from anyspecies, e.g., a mammalian PD-1 protein, a rodent PD-1 protein, aprimate PD-1 protein, a rat PD-1 protein, a mouse PD-1 protein, a pigPD-1 protein, a cow PD-1 protein, a sheep PD-1 protein, a rabbit PD-1protein, a dog PD-1 protein, a human PD-1 protein, etc. Sequences forvarious wild type PD-1 polypeptide sequences (e.g., canine, bovine,sheep, equine, porcine, rodent, mouse, rat, feline, primate, monkey,ape, chimpanzee, and the like) can easily be found and are readilyavailable to one of ordinary skill in the art. For example, the humanPD-1 protein (set forth as SEQ ID NO: 1) is:

Wild type human PD-1 (also known as “programmed cell death 1”, PDCD1, CD279, PD1, SLEB2, hPD-1, hPD-I, and hSLE1) (SEQ ID NO: 1)MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL  bold: transmembranedomain-amino acids 168-191 underline: exemplary native PD-1 mimicpolypeptide-amino acids 26-147

A PD-L protein is a membrane-bound ligand to PD-1. There are two humanPD-L proteins that are referred to in the art as PD-L1 and PD-L2.Example PD-L proteins include those from any species, e.g., a mammalianPD-L protein, a rodent PD-L protein, a primate PD-L protein, a rat PD-Lprotein, a mouse PD-L protein, a pig PD-L protein, a cow PD-L protein, asheep PD-L protein, a rabbit PD-L protein, a dog PD-L protein, a humanPD-L protein, etc. Sequences for various wild type PD-L polypeptidesequences (e.g., canine, bovine, sheep, equine, porcine, rodent, mouse,rat, feline, primate, monkey, ape, chimpanzee, and the like) can easilybe found and are readily available to one of ordinary skill in the art.For example, the human PD-L1 and PD-L2 proteins (set forth as SEQ IDNOs: 36-38, respectively) are:

Wild type human PD-L1(also known as “programmed cell death 1 ligand 1”, PDCD1LG1, CD274, B7-H, B7H1, PDL1, PD-L1, PDCD1L1) (Isoform a)(SEQ ID NO: 36) MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET  (Isoform b) (SEQ ID NO: 37)MRIFAVFIFMTYWHLLNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET  Wild type human PD-L2(also known as “programmed cell death 1 ligand 2”,PDCD1LG2, CD273, B7DC, Btdc, PDL2, PDCD1L2, bA574F11.2) (SEQ ID NO: 38)MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWLLHIFIPFCIIAFIFIATVIALRKQLCQKLYSSKDTTKRPVTTTKREVNSAI 

The transmembrane domain of a wild type PD-1 is readily identifiable. Asan illustrative example, three different domain prediction programs wererun on the wild type human PD-1 protein set forth in SEQ ID NO: 1, andthe following overlapping amino acid regions were determined to define atransmembrane domain: 168-191, 167-189, and 169-191. Thus, atransmembrane domain is present at amino acids 167-191 (e.g., 168-191,167-189, and/or 169-191) of the wild type human PD-1 protein set forthin SEQ ID NO: 1. Thus, in some cases, a PD-1 mimic polypeptide lacksamino acids 167-191, 168-191, 167-189, and/or 169-191 of the wild typehuman PD-1 protein set forth in SEQ ID NO: 1, or the correspondingregion of another wild type PD-1 protein. Sequences for variousadditional wild type PD-1 polypeptide sequences (e.g., canine, bovine,sheep, equine, porcine, rodent, mouse, rat, feline, primate, monkey,ape, chimpanzee, and the like) can easily be found and are readilyavailable to one of ordinary skill in the art.

A suitable PD-1 mimic polypeptide specifically binds to PD-L (e.g.,PD-L1 and/or PD-L2 on a target cell, e.g., on a cancer cell) and therebyreduces (e.g., blocks, prevents, etc.) the interaction between the PD-Land PD-1 (e.g., PD-1 on an immune cell, e.g., on a T cell). Thus, asubject PD-1 mimic polypeptide can be considered to be an engineereddecoy receptor for PD-L (e.g., PD-L1 and/or PD-L2). By reducing theinteraction between PD-L and PD-1, a subject PD-1 mimic polypeptide candecrease the immune inhibitory signals produced by the PD-L/PD-1interaction, and therefore can increase the immune response (e.g., byincreasing T cell activation).

A suitable PD-1 mimic polypeptide comprises the portion of PD-1 that issufficient to bind PD-L1 at a recognizable affinity, e.g., highaffinity, which normally lies between the signal sequence and thetransmembrane domain, or a fragment thereof that retains the bindingactivity. Thus, a subject PD-1 mimic polypeptide can comprise animmunoglobulin domain, or a portion thereof (as described below) that issufficient to bind PD-L1 with a recognizable affinity. A subject PD-1mimic polypeptide (e.g., a high affinity PD-1 mimic polypeptide) cancomprise portions of a wild type PD-1 protein other than theimmunoglobulin domain, including, for example, contiguous amino acids ofany of the sequences set forth in SEQ ID NOs: 2 and 27-34 (or thecorresponding sequences of any other PD-1 protein, e.g., any othermammalian PD-1 protein).

In some cases, the portion of PD-1 that is sufficient to bind PD-L1and/or PD-L2 includes all or a portion of the immunoglobulin domain of awild type PD-1 polypeptide, which can readily be identified by one ofordinary skill in the art. For example, a scan of the wild type humanPD-1 sequence set forth in SEQ ID NO: 1 reveals that the region fromamino acids 35-146 contains an immunoglobulin domain (Table 1). A PD-1mimic polypeptide (e.g., a high affinity PD-1 mimic polypeptide) caninclude all or a portion of the immunoglobulin domain of PD-1; and mayfurther comprise one or more amino acids from PD-1 outside of theimmunoglobulin domain; and may comprise amino acid sequences other thanPD-1, which include without limitation immunoglobulin Fc regionsequences.

TABLE 1 Immunoglobulin domains of wild type human PD-1 identified byvarious sequence analysis software. Amino acids Domain Database 35-145Immunoglobulin-like domain PROSITE 38-128 Immunoglobulin V-set domainPfam 39-145 Immunoglobulin subtype SMART 39-146 Immunoglobulin-like foldGENE3D 49-125 Immunoglobulin V-Type (IGv); SMART Immunoglobulin V-set,subgroup 42-136 IgV; Immunoglobulin variable domain (IgV) NCBI 39-125IG_like; Immunoglobulin like NCBI

In some cases, a PD-1 mimic polypeptide includes an amino acid sequencehaving 85% or more sequence identity (e.g., 90% or more, 95% or more,98% or more, 99% or more, 99.2% or more, 99.5% or more, 99.8% or more,99.9% or more, or 100% sequence identity) to a wild type PD-1polypeptide (e.g., to the corresponding region of a wild type PD-1polypeptide) (e.g., a mammalian wild type PD-1 polypeptide; the humanwild type PD-1 protein set forth in SEQ ID NO: 1, and the like).

In some cases, a PD-1 mimic polypeptide includes an amino acid sequencehaving 85% or more sequence identity (e.g., 90% or more, 95% or more,98% or more, 99% or more, 99.2% or more, 99.5% or more, 99.8% or more,99.9% or more, or 100% sequence identity) to an immunoglobulin domain ofa wild type PD-1 polypeptide (e.g., an Immunoglobulin V-set domain orImmunoglobulin V-Type domain (IGv domain); an Immunoglobulin-like fold;an Immunoglobulin variable domain; an Immunoglobulin like domain, andthe like.). Sequences for various additional wild type PD-1 polypeptidesequences (e.g., canine, bovine, sheep, equine, porcine, rodent, mouse,rat, feline, primate, monkey, ape, chimpanzee, and the like) can easilybe found and are readily available to one of ordinary skill in the art.

In some cases, a PD-1 mimic polypeptide includes an amino acid sequencehaving 85% or more sequence identity (e.g., 90% or more, 95% or more,98% or more, 99% or more, 99.2% or more, 99.5% or more, 99.8% or more,99.9% or more, or 100% sequence identity) to an immunoglobulindomain-containing region of the human PD-1 amino acid sequence set forthin SEQ ID NO: 1; or the corresponding region of another wild type PD-1protein (e.g., another mammalian wild type PD-1 protein). In some cases,a PD-1 mimic polypeptide includes an amino acid sequence having 85% ormore sequence identity (e.g., 90% or more, 95% or more, 98% or more, 99%or more, 99.2% or more, 99.5% or more, 99.8% or more, 99.9% or more, or100% sequence identity) to amino acids 35-145 (SEQ ID NO: 27), 38-128(SEQ ID NO: 28), 39-145 (SEQ ID NO: 29), 39-146 (SEQ ID NO: 30), 49-125(SEQ ID NO: 31), 42-136 (SEQ ID NO: 32), 39-125 (SEQ ID NO: 33), 35-146(SEQ ID NO: 34), 1-166 (SEQ ID NO: 35), and/or 26-147 (SEQ ID NO: 2) ofthe wild type human PD-1 polypeptide amino acid sequence set forth inSEQ ID NO: 1; or the corresponding region of another wild type PD-1protein (e.g., another mammalian wild type PD-1 protein). In some cases,a PD-1 mimic polypeptide includes amino acids 35-145, 38-128, 39-145,39-146, 49-125, 42-136, 39-125, 35-146, 1-166 and/or 26-147 (SEQ ID NO:2) of the wild type human PD-1 protein sequence (amino acid sequence)set forth in SEQ ID NO: 1; or the corresponding region of another wildtype PD-1 protein (e.g., another mammalian wild type PD-1 protein).

In some cases, a PD-1 mimic polypeptide includes an amino acid sequencehaving 85% or more sequence identity (e.g., 90% or more, 95% or more,98% or more, 99% or more, 99.2% or more, 99.5% or more, 99.8% or more,99.9% or more, or 100% sequence identity) to the native PD-1 mimicpolypeptide amino acid sequence set forth in SEQ ID NO: 2 (i.e., thePD-1 protein fragment set forth in SEQ ID NO: 2); or the correspondingregion of another wild type PD-1 protein (e.g., another mammalian wildtype PD-1 protein). The polypeptide set forth in SEQ ID NO: 2 is aprotein fragment (amino acids 26-147) of the wild type human PD-1protein (SEQ ID NO: 1). The amino acid sequence set forth in SEQ ID NO:2 includes an immunoglobulin domain of the wild type human PD-1polypeptide. In some cases, a PD-1 mimic polypeptide includes the aminoacid sequence set forth in SEQ ID NO: 2 (i.e., amino acids 26-147 of thehuman PD-1 protein sequence set forth in SEQ ID NO: 1)(i.e., in somecases, a PD-1 mimic polypeptide includes the PD-1 protein fragment setforth in SEQ ID NO: 2); or the corresponding region of another wild typePD-1 protein, e.g., another mammalian wild type PD-1 protein.

In some cases, a PD-1 mimic polypeptide includes an amino acid sequencehaving 85% or more sequence identity (e.g., 90% or more, 95% or more,98% or more, 99% or more, 99.2% or more, 99.5% or more, 99.8% or more,99.9% or more, or 100% sequence identity) to the amino acid sequence setforth in any of SEQ ID NOs: 2-25 (e.g., 85% or more, 90% or more, 95% ormore, 98% or more, 99% or more, 99.2% or more, 99.5% or more, 99.8% ormore, 99.9% or more, or 100% sequence identity to any of SEQ ID NOs:3-25, SEQ ID NOs: 2-23, SEQ ID NOs: 3-23, SEQ ID NOs: 24-25, etc.). Insome cases, a PD-1 mimic polypeptide includes the amino acid sequenceset forth in any of SEQ ID NOs: 2-25 (e.g., SEQ ID NOs: 3-25, SEQ IDNOs: 2-23, SEQ ID NOs: 3-23, SEQ ID NOs: 24-25, etc.).

A PD-1 mimic polypeptide having no mutations relative to thecorresponding region of a wild type PD-1 protein (i.e., where the PD-1mimic polypeptide is a fragment of a wild type protein) is referred toherein as a “native PD-1 mimic polypeptide.” A native PD-1 mimicpolypeptide can be used as a control in various instances, for example,in some cases when determining whether a PD-1 mimic polypeptide is a“high affinity PD-1 mimic polypeptide.”

High Affinity PD-1 Mimic Polypeptide.

A “high affinity PD-1 mimic polypeptide” is a PD-1 mimic polypeptide (asdefined above, and thus lacks a transmembrane domain of a wild type PD-1protein) that has an amino acid mutation (i.e., an amino acid change)relative to a wild type PD-1 protein, (e.g., relative to thecorresponding region of a wild type PD-1 protein, relative to theectodomain of a wild type PD-1 protein, relative to the immunoglobulindomain of a wild type PD-1 protein, relative to a native PD-1 mimicpolypeptide, etc.), where the amino acid mutation increases the affinityof the PD-1 mimic polypeptide for PD-L1 such that the affinity of thehigh affinity PD-1 mimic polypeptide for PD-L1 is greater than thataffinity of the wild type PD-1 protein (and/or the corresponding nativePD-1 mimic polypeptide) for PD-L1. For example, the amino acid mutationcan increase the affinity by decreasing the off-rate by at least10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least500-fold, or more.

According to the present disclosure, amino acid mutations (i.e.,changes) include any naturally occurring or man-made amino acidmodifications known or later discovered in the field. In someembodiments, amino acid changes include, e.g., substitution, deletion,addition, insertion, etc. of one or more amino acids. In someembodiments, amino acid changes include replacing an existing amino acidwith another amino acid. In related embodiments, amino acid changesinclude replacing one or more existing amino acids with non-naturalamino acids, or inserting one or more non-natural amino acids. Aminoacid changes may be made in 1 or more (e.g, 2 or more, 3 or more, 4 ormore, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more,11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more,17 or more, 18 or more, 19 or more, 20 or more, etc.) amino acidresidues relative to a wild type sequence. The one or more amino acidchanges can confer various properties to the high affinity PD-1 mimicpolypeptide, e.g., affecting the stability, binding activity and/orspecificity, etc.

In some cases, a high affinity PD-1 mimic polypeptide includes an aminoacid change (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 ormore, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 ormore, 18 or more, 19 or more, or 20 amino acid changes) relative to awild type PD-1 polypeptide (e.g., relative to the corresponding regionof a wild type PD-1 polypeptide) (e.g., a mammalian wild type PD-1polypeptide; the human wild type PD-1 protein set forth in SEQ ID NO: 1,and the like).

In some cases, a high affinity PD-1 mimic polypeptide includes an aminoacid change (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 ormore, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 ormore, 18 or more, 19 or more, or 20 amino acid changes) relative to theimmunoglobulin domain of a wild type PD-1 polypeptide (e.g., amino acids35-145, 38-128, 39-145, 39-146, 49-125, 42-136, 39-125, 35-146, 1-166and/or 26-147 (SEQ ID NO: 2) of the wild type human PD-1 polypeptideamino acid sequence set forth in SEQ ID NO: 1; or the correspondingregion of another wild type PD-1 protein, e.g., another mammalian wildtype PD-1 protein). In some cases, a high affinity PD-1 mimicpolypeptide includes an amino acid change (e.g., 1 or more, 2 or more, 3or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 ormore, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 ormore, 16 or more, 17 or more, 18 or more, 19 or more, or 20 amino acidchanges) relative to the ectodomain of a wild type PD-1 polypeptide.

In some cases, a high affinity PD-1 mimic polypeptide includes an aminoacid change (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 ormore, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 ormore, 18 or more, 19 or more, or 20 amino acid changes) relative to theamino acid sequence set forth in SEQ ID NO: 2 (which is a human “nativePD-1 mimic polypeptide”, as defined above); or the corresponding regionof another wild type PD-1 protein, e.g., another mammalian wild typePD-1 protein. In some cases, a high affinity PD-1 mimic polypeptideincludes an amino acid change (e.g., 1 or more, 2 or more, 3 or more, 4or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 ormore, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 ormore, 17 or more, 18 or more, 19 or more, or 20 amino acid changes)relative to the amino acid sequence set forth in any of SEQ ID NOs: 2-25(e.g., relative to any of SEQ ID NOs: 3-25, SEQ ID NOs: 2-23, SEQ IDNOs: 3-23, SEQ ID NOs: 24-25, etc.).

In some cases, a high affinity PD-1 mimic polypeptide includes an aminoacid change (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 ormore, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 ormore, 18 or more, 19 or more, or 20 amino acid changes) relative to awild type PD-1 polypeptide (e.g., to the corresponding region of a wildtype PD-1 polypeptide) (e.g., a mammalian wild type PD-1 polypeptide;the human wild type PD-1 protein set forth in SEQ ID NO: 1, and thelike), and includes an amino acid sequence having 85% or more sequenceidentity (e.g., 90% or more, 95% or more, 98% or more, 99% or more,99.2% or more, 99.5% or more, 99.8% or more, 99.9% or more, or 100%sequence identity) to a wild type PD-1 polypeptide (e.g., to thecorresponding region of a wild type PD-1 polypeptide) (e.g., a mammalianwild type PD-1 polypeptide; the human wild type PD-1 protein set forthin SEQ ID NO: 1, and the like).

In some cases, a high affinity PD-1 mimic polypeptide includes an aminoacid change (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 ormore, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 ormore, 18 or more, 19 or more, or 20 amino acid changes) relative to theimmunoglobulin domain of a wild type PD-1 polypeptide; or thecorresponding region of another wild type PD-1 protein, e.g., anothermammalian wild type PD-1 protein, and includes an amino acid sequencehaving 85% or more sequence identity (e.g., 90% or more, 95% or more,98% or more, 99% or more, 99.2% or more, 99.5% or more, 99.8% or more,99.9% or more, or 100% sequence identity) to the immunoglobulin domainof a wild type PD-1 polypeptide (e.g., amino acids 35-145, 38-128,39-145, 39-146, 49-125, 42-136, 39-125, or 35-146 of the wild type humanPD-1 polypeptide amino acid sequence set forth in SEQ ID NO: 1); or thecorresponding region of another wild type PD-1 protein, e.g., anothermammalian wild type PD-1 protein.

In some cases, a high affinity PD-1 mimic polypeptide includes an aminoacid change (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 ormore, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 ormore, 18 or more, 19 or more, or 20 amino acid changes) relative toamino acids 35-145, 38-128, 39-145, 39-146, 49-125, 42-136, 39-125,35-146, 1-166 and/or 26-147 (SEQ ID NO: 2) of the wild type human PD-1polypeptide amino acid sequence set forth in SEQ ID NO: 1; or thecorresponding region of another wild type PD-1 protein, e.g., anothermammalian wild type PD-1 protein; and includes an amino acid sequencehaving 85% or more sequence identity (e.g., 90% or more, 95% or more,98% or more, 99% or more, 99.2% or more, 99.5% or more, 99.8% or more,99.9% or more, or 100% sequence identity) to amino acids 35-145, 38-128,39-145, 39-146, 49-125, 42-136, 39-125, 35-146, 1-166 and/or 26-147 (SEQID NO: 2) of the wild type human PD-1 polypeptide amino acid sequenceset forth in SEQ ID NO: 1; or the corresponding region of another wildtype PD-1 protein, e.g., another mammalian wild type PD-1 protein.

In some cases, a high affinity PD-1 mimic polypeptide includes an aminoacid change (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 ormore, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 ormore, 18 or more, 19 or more, or 20 amino acid changes) relative to theamino acid sequence set forth in SEQ ID NO: 2; or the correspondingregion of another wild type PD-1 protein, e.g., another mammalian wildtype PD-1 protein; and includes an amino acid sequence having 85% ormore sequence identity (e.g., 90% or more, 95% or more, 98% or more, 99%or more, 99.2% or more, 99.5% or more, 99.8% or more, 99.9% or more, or100% sequence identity) to the amino acid sequence set forth in SEQ IDNO: 2; or the corresponding region of another wild type PD-1 protein,e.g., another mammalian wild type PD-1 protein.

In some cases, a high affinity PD-1 mimic polypeptide includes an aminoacid change (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 ormore, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 ormore, 18 or more, 19 or more, or 20 amino acid changes) relative to theamino acid sequence set forth in any of SEQ ID NOs: 2-25 (e.g., relativeto any of SEQ ID NOs: 3-25, SEQ ID NOs: 2-23, SEQ ID NOs: 3-23, SEQ IDNOs: 24-25, etc.); and includes an amino acid sequence having 85% ormore sequence identity (e.g., 90% or more, 95% or more, 98% or more, 99%or more, 99.2% or more, 99.5% or more, 99.8% or more, 99.9% or more, or100% sequence identity) to the amino acid sequence set forth in any ofSEQ ID NOs: 2-25 (e.g., 85% or more, 90% or more, 95% or more, 98% ormore, 99% or more, 99.2% or more, 99.5% or more, 99.8% or more, 99.9% ormore, or 100% sequence identity to any of SEQ ID NOs: 3-25, SEQ ID NOs:2-23, SEQ ID NOs: 3-23, SEQ ID NOs: 24-25, etc.).

In some cases, a high affinity PD-1 mimic polypeptide of the disclosureincludes one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more,6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18or more, 14 or more, 15 or more) amino acid changes located at aminoacid positions of PD-1 that contacts PD-L1. For example, in some cases,the one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 ormore, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 ormore, 14 or more, 15 or more) amino acid changes is located at an aminoacid position, relative to the protein fragment (of human wild typePD-1) set forth in SEQ ID NO: 2 (which is a protein fragment of thehuman wild type PD-1 protein), selected from: V39, N41, Y43, M45, S48,N49, Q50, T51, D52, K53, A56, Q63, G65, Q66, L97, S102, L103, A104,P105, K106, and A107; or the corresponding amino acid position relativeto another wild type PD-1 protein. For example, refer to FIG. 3.

In some cases, a high affinity PD-1 mimic polypeptide of the disclosureincludes one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more,6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18or more, 14 or more, 15 or more) amino acid changes located at an aminoacid position, relative to the protein fragment (of human wild typePD-1) set forth in SEQ ID NO: 2 (which is a protein fragment of thehuman wild type PD-1 protein), selected from: V39, L40, N41, Y43, R44,M45, S48, N49, Q50, T51, D52, K53, A56, Q63, G65, Q66, V72, H82, M83,R90, Y96, L97, A100, S102, L103, A104, P105, K106, and A107; or thecorresponding amino acid position relative to another wild type PD-1protein. For example, refer to FIG. 3.

In some cases, a high affinity PD-1 mimic polypeptide of the disclosureincludes amino acid changes located at amino acid positions, relative tothe protein fragment (of human wild type PD-1) set forth in SEQ ID NO: 2(which is a protein fragment of the human wild type PD-1 protein),selected from: (a) V39, N41, Y43, M45, S48, N49, Q50, K53, A56, Q63,G65, Q66, L97, S102, L103, A104, K106, and A107, or the correspondingamino acid positions relative to another wild type PD-1 protein; (b)V39, N41, Y43, M45, S48, Q50, T51, D52F, K53, A56, Q63, G65, Q66, L97,S102, L103, A104, K106, and A107, or the corresponding amino acidpositions relative to another wild type PD-1 protein; (c) V39, L40, N41,Y43, R44, M45, N49, K53, M83, L97, A100, and A107, or the correspondingamino acid positions relative to another wild type PD-1 protein; (d)V39, L40, N41, Y43, M45, N49, K53, Q66P, M83, L97, and A107, or thecorresponding amino acid positions relative to another wild type PD-1protein; (e) V39, L40, N41, Y43, M45, N49, K53, Q66P, H82, M83, L97,A100, and A107, or the corresponding amino acid positions relative toanother wild type PD-1 protein; (f) V39, L40, N41, Y43, M45, N49, K53,M83, L97, A100, and A107, or the corresponding amino acid positionsrelative to another wild type PD-1 protein; (g) V39, L40, N41, Y43, R44,M45, N49, K53, L97, A100, and A107, or the corresponding amino acidpositions relative to another wild type PD-1 protein; and (h) V39, L40,N41, Y43, M45, N49, K53, L97, A100, and A107, or the corresponding aminoacid positions relative to another wild type PD-1 protein. For example,refer to FIG. 3.

In some cases, a high affinity PD-1 mimic polypeptide of the disclosureincludes one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more,6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18or more, 14 or more, 15 or more) amino acid changes, relative to theprotein fragment (of human wild type PD-1) set forth in SEQ ID NO: 2(which is a protein fragment of the human wild type PD-1 protein),selected from: (1) V39H or V39R; (2) L40V or L40I; (3) N41I or N41V; (4)Y43F or Y43H; (5) R44Y or R44L; (6) M45Q, M45E, M45L, or M45D; (7) S48D,S48L, S48N, S48G, or S48V; (8) N49C, N49G, N49Y, or N49S; (9) Q50K,Q50E, or Q50H; (10) T51V, T51L, or T51A; (11) D52F, D52R, D52Y, or D52V;(12) K53T or K53L; (13) A56S or A56L; (14) Q63T, Q63I, Q63E, Q63L, orQ63P; (15) G65N, G65R, G65I, G65L, G65F, or G65V; (16) Q66P; (17) V72I;(18) H82Q; (19) M83L or M83F; (20) R90K; (21) Y96F; (22) L97Y, L97V, orL97I; (23) A100I or A100V; (24) S102T or S102A; (25) L103I, L103Y, orL103F; (26) A104S, A104H, or A104D; (27) P105A; (28) K106G, K106E,K106I, K106V, K106R, or K106T; and (29) A107P, A107I, or A107V; or achange that results in the same amino acid at the corresponding positionrelative to another wild type PD-1 protein. For example, refer to FIG.3.

In some cases, a high affinity PD-1 mimic polypeptide of the disclosureincludes amino acid changes, relative to the protein fragment (of humanwild type PD-1) set forth in SEQ ID NO: 2 (which is a protein fragmentof the human wild type PD-1 protein), selected from:

(a) {V39H or V39R}, {N41I or N41V}, {Y43F or Y43H}, {M45Q, M45E, M45L,or M45D}, {S48D, S48L, S48N, S48G, or S48V}, {N49C, N49G, N49Y, orN49S}, {Q50K, Q50E, or Q50H}, {K53T or K53L}, {A56S or A56L}, {Q63T,Q63I, Q63E, Q63L, or Q63P}, {G65N, G65R, G65I, G65L, G65F, or G65V},{Q66P}, {L97Y, L97V, or L97I}, {S102T or S102A}, {L103I, L103Y, orL103F}, {A104S, A104H, or A104D}, {K106G, K106E, K106I, K106V, K106R, orK106T}, and {A107P, A107I, or A107V}; or changes that result in the sameamino acids at the corresponding positions relative to another wild typePD-1 protein;

(b) {V39H or V39R}, {N41I or N41V}, {Y43F or Y43H}, {M45Q, M45E, M45L,or M45D}, {S48D, S48L, S48N, S48G, or S48V}, {Q50K, Q50E, or Q50H},{T51V, T51L, or T51A}, {D52F, D52R, D52Y, or D52V}, {K53T or K53L},{A56S or A56L}, {Q63T, Q63I, Q63E, Q63L, or Q63P}, {G65N, G65R, G65I,G65L, G65F, or G65V}, {Q66P}, {L97Y, L97V, or L97I}, {S102T or S102A},{L103I, L103Y, or L103F}, {A104S, A104H, or A104D}, {K106G, K106E,K106I, K106V, K106R, or K106T}, and {A107P, A107I, or A107V}; or changesthat result in the same amino acids at the corresponding positionsrelative to another wild type PD-1 protein;

-   -   (c){V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43F or        Y43H}, {R44Y or R44L}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G,        N49Y, or N49S}, {K53T or K53L}, {M83L or M83F}, {L97Y, L97V, or        L97I}, {A100I or A100V}, and {A107P, A107I, or A107V}; or        changes that result in the same amino acids at the corresponding        positions relative to another wild type PD-1 protein;    -   (d) {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43F or        Y43H}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, or N49S},        {K53T or K53L}, {Q66P}, {M83L or M83F}, {L97Y, L97V, or L97I},        and {A107P, A107I, or A107V}; or changes that result in the same        amino acids at the corresponding positions relative to another        wild type PD-1 protein;

(e) {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43F or Y43H},{M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, or N49S}, {K53T orK53L}, {Q66P}, {H82Q}, {M83L or M83F}, {L97Y, L97V, or L97I}, {A100I orA100V}, and {A107P, A107I, or A107V}; or changes that result in the sameamino acids at the corresponding positions relative to another wild typePD-1 protein;

(f) {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43F or Y43H},{M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, or N49S}, {K53T orK53L}, {M83L or M83F}, {L97Y, L97V, or L97I}, {A100I or A100V}, and{A107P, A107I, or A107V}; or changes that result in the same amino acidsat the corresponding positions relative to another wild type PD-1protein;

(g) {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43F or Y43H},{R44Y or R44L}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, orN49S}, {K53T or K53L}, {L97Y, L97V, or L97I}, {A100I or A100V}, and{A107P, A107I, or A107V}; or changes that result in the same amino acidsat the corresponding positions relative to another wild type PD-1protein; and (h) {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43For Y43H}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, or N49S},{K53T or K53L}, {L97Y, L97V, or L97I}, {A100I or A100V}, and {A107P,A107I, or A107V}; or changes that result in the same amino acids at thecorresponding positions relative to another wild type PD-1 protein. Forexample, refer to FIG. 3.

In some cases, a high affinity PD-1 mimic polypeptide of the disclosureincludes amino acid changes, relative to the protein fragment (of humanwild type PD-1) set forth in SEQ ID NO: 2 (which is a protein fragmentof the human wild type PD-1 protein), selected from:

(a) V39R, N41V, Y43H, M45E, S48G, N49Y, Q50E, K53T, A56S, Q63T, G65L,Q66P, L97V, S102A, L103F, A104H, K106V, and A107I; or changes thatresult in the same amino acids at the corresponding positions relativeto another wild type PD-1 protein;

(b) V39R, N41V, Y43H, M45E, S48N, Q50H, T51A, D52Y, K53T, A56L, Q63L,G65F, Q66P, L97I, S102T, L103F, A104D, K106R, and A107I; or changes thatresult in the same amino acids at the corresponding positions relativeto another wild type PD-1 protein;

(c) V39H, L40V, N41V, Y43H, R44Y, M45E, N49G, K53T, M83L, L97V, A100I,and A107I; or changes that result in the same amino acids at thecorresponding positions relative to another wild type PD-1 protein;

(d) V39H, L40V, N41V, Y43H, M45E, N49G, K53T, Q66P, M83L, L97V, andA107I; or changes that result in the same amino acids at thecorresponding positions relative to another wild type PD-1 protein;

-   -   (e) V39H, L40V, N41V, Y43H, M45E, N49S, K53T, Q66P, H82Q, M83L,        L97V, A100V, and A107I; or changes that result in the same amino        acids at the corresponding positions relative to another wild        type PD-1 protein;

(f) V39H, L40I, N41I, Y43H, M45E, N49G, K53T, M83L, L97V, A100V, andA107I; or changes that result in the same amino acids at thecorresponding positions relative to another wild type PD-1 protein;

(g) V39H, L40V, N41I, Y43H, R44L, M45E, N49G, K53T, L97V, A100V, andA107I; or changes that result in the same amino acids at thecorresponding positions relative to another wild type PD-1 protein;

(h) V39H, L40V, N41I, Y43H, M45E, N49G, K53T, L97V, A100V, and A107I; orchanges that result in the same amino acids at the correspondingpositions relative to another wild type PD-1 protein; and

-   -   (i) V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, A100V, and        A107I or changes that result in the same amino acids at the        corresponding positions relative to another wild type PD-1        protein. For example, refer to FIG. 3.

Affinity

As subject high affinity PD-1 mimic polypeptide has an increasedaffinity for PD-L1 as compared to the affinity for PD-L1 of a wild typePD-1 protein, and/or as compared to the affinity for PD-L1 of a PD-1mimic polypeptide that does not have an amino acid change relative to acorresponding sequence of a wild type PD-1 polypeptide (e.g., a nativePD-1 mimic polypeptide, as defined above).

In some embodiments, the high affinity PD-1 mimic polypeptide has aK_(d) of 1×10⁻⁷ M or less (e.g., 10⁻⁸ M or less, 10⁻⁹ M or less, 10⁻¹⁰ Mor less, 10⁻¹¹ M or less, 10⁻¹² M or less, 10⁻¹³ M or less, 10⁻¹⁴ M orless, 10⁻¹⁵ M or less, or 10⁻¹⁶ M or less) for PD-L1. In some cases, thehigh affinity PD-1 mimic polypeptide has an affinity for PD-L1 in arange of from 1 fM to 1 μM (e.g., from 1 fM to 800 nM, from 10 fM to 500nM, from 100 fM to 100 nM, from 500 fM to 50 nM, from 800 fM to 50 nM,from 1 pM to 50 nM, from 10 pM to 50 nM, from 50 pM to 50 nM, from 100pM to 50 nM, from 500 fM to 100 nM, from 800 fM to 100 nM, from 1 pM to100 nM, from 10 pM to 100 nM, from 50 pM to 100 nM, or from 100 pM to100 nM). In some cases, the high affinity PD-1 mimic polypeptide bindsto PD-L1 with an affinity of 1 pM or greater (e.g., 800 nM or greater,500 nM or greater, 200 nM or greater, 100 nM or greater, 50 nM orgreater, 10 nM or greater, 1 nM or greater, 900 pM or greater, 750 pM orgreater, 500 pM or greater, 200 pM or greater, 100 pM or greater, 10 pMor greater, 1 pM or greater, etc.) (where the affinity increases withdecreasing values).

In some embodiments, the high affinity PD-1 mimic polypeptide has anaffinity for PD-L1 that is 2-fold or more (e.g., 5-fold or more, 10-foldor more, 100-fold or more, 500-fold or more, 1000-fold or more,5000-fold or more, 10⁴-fold or more, 10⁵-fold or more, 10⁶-fold or more,10⁷-fold or more, 10⁸-fold or more, etc.) greater than the affinity forPD-L1 of a wild type PD-1 protein; and/or 2-fold or more (e.g., 5-foldor more, 10-fold or more, 100-fold or more, 500-fold or more, 1000-foldor more, 5000-fold or more, 10⁴-fold or more, 10⁵-fold or more, 10⁶-foldor more, 10⁷-fold or more, 10⁸-fold or more, etc.) greater than theaffinity for PD-L1 of a PD-1 mimic polypeptide that does not have anamino acid change relative to a corresponding sequence of a wild typePD-1 polypeptide (e.g., a native PD-1 mimic polypeptide, as definedabove).

In some embodiments, the high affinity PD-1 mimic polypeptide has adissociation half-life for PD-L1 that is 2-fold or more (e.g., 5-fold ormore, 10-fold or more, 100-fold or more, 500-fold or more, 1000-fold ormore, 5000-fold or more, 10⁴-fold or more, 10⁵-fold or more, 10⁶-fold ormore, 10⁷-fold or more, 10⁸-fold or more, etc.) greater than thedissociation half-life for PD-L1 of a wild type PD-1 protein; and/or2-fold or more (e.g., 5-fold or more, 10-fold or more, 100-fold or more,500-fold or more, 1000-fold or more, 5000-fold or more, 10⁴-fold ormore, 10⁵-fold or more, 10⁶-fold or more, 10⁷-fold or more, 10⁸-fold ormore, etc.) greater than the dissociation half-life for PD-L1 of a PD-1mimic polypeptide that does not have an amino acid change relative to acorresponding sequence of a wild type PD-1 polypeptide (e.g., a nativePD-1 mimic polypeptide, as defined above). For example, in some cases, anative PD-1 mimic polypeptide (as defined above) has a dissociationhalf-life for PD-L1 of less than 1 second, while a subject high affinityPD-1 mimic polypeptide can have a dissociation half-life of 5 seconds ormore (e.g., 30 seconds or more, 1 minute or more, 5 minutes or more, 10minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes ormore, etc.). For example, the amino acid mutation of a subject highaffinity PD-1 mimic polypeptide can increase the affinity by decreasingthe off-rate by at least 10-fold, at least 20-fold, at least 50-fold, atleast 100-fold, at least 500-fold, or more.

In some cases, a subject high affinity PD-1 mimic polypeptide has adecreased affinity for PD-L2 as compared to the affinity for PD-L2 of acorresponding PD-1 mimic polypeptide that does not have an amino acidchange relative to the wild type PD-1 polypeptide (e.g., a decreasedaffinity for PD-L2 as compared to the affinity for PD-L2 of acorresponding native PD-1 mimic polypeptide, as defined above).

In some cases, a subject high affinity PD-1 mimic polypeptide has agreater affinity for PD-L1 than for PD-L2. For example, in some cases,subject high affinity PD-1 mimic polypeptide specifically binds toPD-L1, but not PD-L2. In some cases, a subject high affinity PD-1 mimicpolypeptide has an affinity for PD-L2 that is characterized by a K_(d)(dissociation constant) that is 2-fold or more (e.g., 5-fold or more,10-fold or more, 100-fold or more, 500-fold or more, 1000-fold or more,5000-fold or more, 10⁴-fold or more, 10⁵-fold or more, 10⁶-fold or more,10⁷-fold or more, 10⁸-fold or more, etc.) greater than the K_(d) thatcharacterizes the affinity of the high affinity PD-1 mimic polypeptidefor PD-L1. In other words, in some cases, the affinity of a subject highaffinity PD-1 mimic polypeptide for PD-L1 can be 2-fold or more (e.g.,5-fold or more, 10-fold or more, 100-fold or more, 500-fold or more,1000-fold or more, 5000-fold or more, 10⁴-fold or more, 10⁵-fold ormore, 10⁶-fold or more, 10⁷-fold or more, 10⁸-fold or more, etc.)greater than the affinity of the subject high affinity PD-1 mimicpolypeptide for PD-L2.

The affinity to bind to PD-L1 and/or PD-L2 can be determined, forexample, by the ability of a high-affinity PD-1 mimic polypeptide tobind to PD-L1 and/or PD-L2 coated on an assay plate; displayed on amicrobial cell surface; in solution; etc. The binding activity ofhigh-affinity PD-1 mimic polypeptides of the present disclosure to PD-L1and/or PD-L2 can be assayed by immobilizing the ligand (e.g., PD-L1and/or PD-L2) or the high-affinity PD-1 mimic polypeptide, to a bead,substrate, cell, etc. Agents can be added in an appropriate buffer andthe binding partners incubated for a period of time at a giventemperature. After washes to remove unbound material, the bound proteincan be released with, for example, SDS, buffers with a high pH, and thelike and analyzed. For example, see FIG. 4 which depicts Surface PlasmonResonance (SPR) plots (i.e., results from SPR experiments that testedthe ability of a high affinity PD-1 mimic polypeptide to bind to PD-L1).

Binding can also be determined by, for example, measuring the ability ofa unlabeled high affinity PD-1 mimic polypeptide to compete with alabeled PD-1 polypeptide (e.g., a labeled native PD-1 mimic polypeptide,as defined above) for binding to PD-L1 (for examples, see FIGS. 5A-5Cand FIGS. 6A-6B). Accordingly, relative binding can be assessed bycomparing the results using a candidate unlabeled high-affinity PD-1mimic polypeptide to results using an unlabeled native PD-1 mimicpolypeptide (as defined above, a PD-1 mimic polypeptide that does nothave an amino acid change relative to the corresponding sequence of awild type PD-1).

Any convenient method can be used to generate a subject high-affinityPD-1 mimic polypeptide. As one example non-limiting example, mutagenesiscan be performed (beginning with a native PD-1 mimic polypeptide, orbeginning with a high-affinity PD-1 mimic polypeptide for the purpose ofgenerating a polypeptide with even greater affinity) to generatecollections of mutated PD-1 mimic polypeptides. Mutagenesis can betargeted to produce changes at particular amino acids, or mutagenesiscan be random. The mutated PD-1 mimic polypeptides can then be screenfor their ability to bind a PD-L protein (e.g., PD-L1 and/or PD-L2). Forexample, a PD-L protein (or a variant of a PD-L protein, e.g., a versionlacking a transmembrane domain) can be labeled (e.g., with a directlabel such as a radioisotope, a fluorescent moiety, etc.; or with anindirect label such as an antigen, an affinity tag, biotin, etc.) andthen can be used to contact the candidate high-affinity PD-1 mimicpolypeptides (e.g., where the candidate high-affinity PD-1 mimicpolypeptides can be attached to a solid surface or displayed on themembrane of a cell, e.g., a yeast cell). By varying the concentration ofPD-L used, one can identify high-affinity PD-1 mimic polypeptides fromamong the candidates (i.e., from among the collection of mutated PD-1mimic polypeptides). See FIGS. 2A-2B for a non-limiting example of howone can identify a subject high-affinity PD-1 mimic polypeptide.

In some embodiments, a high-affinity PD-1 mimic polypeptide of thepresent disclosure is a fusion protein, e.g., fused in frame with asecond polypeptide (a fusion partner). In some embodiments, the secondpolypeptide is capable of increasing the size of the fusion protein,e.g., so that the fusion protein will not be cleared from thecirculation rapidly. As tissue penetration (i.e., the ability topenetrate tissues) can be a distinct advantage of using a subjecthigh-affinity PD-1 mimic polypeptide due to its relatively small size(e.g., compared to a much larger protein such as an antibody, e.g., ananti-PD-1 or anti-PD-L antibody), in some cases, a high affinity PD-1mimic polypeptide is not fused to a second polypeptide, or is fused to asecond polypeptide that is small enough so as not to limit the tissuepenetration of the high affinity PD-1 mimic polypeptide to anunacceptable level (which would depend on the context of the particularmethod and/or desired outcome). Thus, in some cases, the secondpolypeptide (i.e., the polypeptide to which a subject high-affinity PD-1mimic polypeptide is fused) is 200 amino acids or less (e.g., 190 aminoacids or less, 180 amino acids or less, 170 amino acids or less, 160amino acids or less, 150 amino acids or less, 140 amino acids or less,130 amino acids or less, 120 amino acids or less, 110 amino acids orless, 100 amino acids or less, 90 amino acids or less, 80 amino acids orless, 70 amino acids or less, 60 amino acids or less, 50 amino acids orless, 40 amino acids or less, or 30 amino acids or less). In some cases,the fusion protein has a molecular weight average of 200 kD or less, 150kD or less, 100 kD or less, 90 kD or less, 80 kD or less, 70 kD or less,60 kD or less, 50 kD or less, 40 kD or less, or 30 kD or less.

In some embodiments, the second polypeptide is part or whole of animmunoglobulin Fc region (e.g, a human immunoglobulin Fc region) (e.g.,an antibody Fc sequence, a CH3 domain, and the like). In otherembodiments, the second polypeptide is any suitable polypeptide that issubstantially similar to an Fc, e.g., providing increased size,multimerization domains, and/or additional binding or interaction withIg molecules. These fusion proteins can facilitate purification,multimerization, and show an increased half-life in vivo. Fusionproteins having disulfide-linked multimeric structures can also be moreefficient in binding and neutralizing other molecules than a monomerichigh-affinity PD-1 mimic polypeptide.

When fused to a heterologous polypeptide, the portion corresponding tothe high affinity PD-1 mimic polypeptide can be referred to as the “highaffinity PD-1 mimic polypeptide portion.” High affinity PD-1 mimicpolypeptides (e.g., the high affinity PD-1 mimic polypeptide portion)can be 70 amino acids or more in length (e.g., 75 amino acids or more,80 amino acids or more, 85 amino acids or more, 90 amino acids or more,95 amino acids or more, 100 amino acids or more, 105 amino acids ormore, 110 amino acids or more, 115 amino acids or more, 120 amino acidsor more, 125 amino acids or more, or 130 amino acids or more), up to thefull-length of the portion of the wild-type protein that is N-terminalto the transmembrane domain (e.g., 165-170 amino acids for the humanPD-1 protein), and can further be fused to a heterologous polypeptide,e.g., an immunoglobulin Fc. In some cases, a high affinity PD-1 mimicpolypeptide (e.g., the high affinity PD-1 mimic polypeptide portion) hasa length in a range of from 70 amino acids to 170 amino acids (e.g.,from 70 amino acids to 166 amino acids, from 75 amino acids to 170 aminoacids, from 80 amino acids to 170 amino acids, etc.).

In some embodiments, a high affinity PD-1 mimic polypeptide is fused orotherwise joined to an immunoglobulin sequence to form a chimericprotein. The immunoglobulin sequence can be an immunoglobulin constantdomain(s). The immunoglobulin moiety in such chimeras may be obtainedfrom any species, usually human, and includes IgG1, IgG2, IgG3 or IgG4subtypes, IgA, IgE, IgD or IgM. The immunoglobulin moiety may compriseone or more domains, e.g., CH1, CH2, CH3, etc.

Chimeras constructed from a sequence linked to an appropriateimmunoglobulin constant domain sequence are known in the art. In suchfusions, the encoded chimeric polypeptide may retain at leastfunctionally active hinge, CH2 and CH3 domains of the constant region ofan immunoglobulin heavy chain. Fusions are also made to the C-terminusof the Fc portion of a constant domain, or immediately N-terminal to theCH1 of the heavy chain or the corresponding region of the light chain.The precise site at which the fusion is made is not critical; particularsites are well known and may be selected in order to optimize thebiological activity, secretion or binding characteristics of the highaffinity PD-1 mimic polypeptide:immunoglobulin chimeras. In someembodiments, the high affinity PD-1 mimic polypeptide:immunoglobulinchimeras are assembled as monomers, or hetero- or homo-multimers, and insome cases as dimers or tetramers.

Although the presence of an immunoglobulin light chain is not required,an immunoglobulin light chain may be included, either covalentlyassociated to a high affinity PD-1 mimic polypeptide:immunoglobulinheavy chain fusion polypeptide, or directly fused to the polypeptide. Asingle chain construct may be used to provide both heavy and light chainconstant regions.

In other fusion protein constructs, the second polypeptide is a markersequence, such as a peptide that facilitates purification of the fusedpolypeptide. For example, the marker amino acid sequence can be ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Another peptide tag useful for purification, the “HA” tag,corresponds to an epitope derived from the influenza hemagglutininprotein. Wilson et al., Cell 37: 767, 1984. The addition of peptidemoieties to facilitate handling of polypeptides are familiar and routinetechniques in the art.

In some cases, a subject high affinity PD-1 mimic polypeptide ismultivalent (e.g., tetravalent).

High affinity PD-1 mimic polypeptides can be monomeric or multimeric,i.e. dimer, trimer, tetramer, etc. For example, one or more PD-L1(and/or PD-L2) binding domains can be covalently or non-covalentlylinked, e.g., as a fusion protein; disulfide bonded; through biotinbinding to avidin, streptavidin, etc. Such monomeric or multimerichigh-affinity PD-1 mimic polypeptides are useful as single agents tostimulate an immune response (e.g., to stimulate a general immuneresponse and/or to stimulate a response directed to a cell expressingPD-L1, e.g., a cancer cell); or in combination with other bindingagents, e.g., monoclonal antibodies.

For example, a subject high affinity PD-1 mimic polypeptide can have afusion partner where the fusion partner provides a multimerizationdomain (i.e., a domain that facilitates multimerization, e.g., a domainthe facilitates dimerization). For example, a fusion partner can be anyconvenient protein-protein interaction domain (e.g., a leucine zippermotif, a synzip polypeptide (a polypeptide pair), a CH 3 domain, and thelike).

High affinity PD-1 mimic polypeptides of the present disclosure can bemodified, e.g., joined to a wide variety of other oligopeptides orproteins for a variety of purposes. For example, post-translationallymodified, for example by prenylation, acetylation, amidation,carboxylation, glycosylation, PEGylation (covalent attachment ofpolyethylene glycol (PEG) polymer chains), etc. Such modifications canalso include modifications of glycosylation, e.g., those made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing or in further processing steps; e.g., byexposing the polypeptide to enzymes which affect glycosylation, such asmammalian glycosylating or deglycosylating enzymes. In some embodiments,a subject high affinity PD-1 mimic polypeptide has one or morephosphorylated amino acid residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine.

In some other embodiments, high affinity PD-1 mimic polypeptides of thedisclosure include reagents further modified to improve their resistanceto proteolytic degradation or to optimize solubility properties or torender them more suitable as a therapeutic agent. For example, variantsof the present disclosure further include analogs containing residuesother than naturally occurring L-amino acids, e.g., D-amino acids ornon-naturally occurring synthetic amino acids. D-amino acids may besubstituted for some or all of the amino acid residues.

In addition to use as a treatment for various disorders and diseases,high affinity PD-1 mimic polypeptides are useful, for example, as anadjuvant to increase immune function, (e.g., when combined with aspecific binding agent, e.g., an antibody, in some cases to a tumor cellspecific antibody as defined herein)(e.g., by stimulating, or reducinginhibition, of a number of immune cells, such as macrophages, dendriticcells, neutrophils, granulocytes, antigen presenting cells, T cells, andthe like).

High affinity PD-1 mimic polypeptides are also useful as imaging agents,e.g., when conjugated to a detectable label, which can be used forvarious purposes, e.g., as diagnostic reagents. For example, in somecases a subject method is a method of diagnosing or prognosing cancer inan individual (e.g., a cancer in which the presence, level, and/orlocation of detectable PD-L1 can be diagnostic and/or prognostic).

In some embodiments of the disclosure, the high affinity PD-1 mimicpolypeptide is coupled or conjugated to one or more imaging moieties,i.e. a detectable label. As used herein, “cytotoxic moiety” refers to amoiety that inhibits cell growth or promotes cell death when proximateto or absorbed by the cell. Suitable cytotoxic moieties in this regardinclude radioactive isotopes (radionuclides), chemotoxic agents such asdifferentiation inducers and small chemotoxic drugs, toxin proteins, andderivatives thereof.

As utilized herein, “imaging moiety”, or detectable label, refers to amoiety that can be utilized to increase contrast between a tumor and thesurrounding healthy tissue in a visualization technique, e.g.,radiography, positron-emission tomography (PET), magnetic resonanceimaging (MRI), direct or indirect visual inspection. Thus, suitableimaging moieties include radiography moieties, e.g., heavy metals andradiation emitting moieties, positron emitting moieties, magneticresonance contrast moieties, and optically visible moieties (e.g.,fluorescent or visible-spectrum dyes, visible particles, etc. It will beappreciated by one of ordinary skill that some overlap exists betweenwhat is a therapeutic moiety and what is an imaging moiety.

In general, therapeutic or imaging agents can be conjugated to a highaffinity PD-1 mimic polypeptide moiety by any suitable technique, withappropriate consideration of the need for pharmacokinetic stability andreduced overall toxicity to the patient. A direct reaction between anagent and PD-L1 is possible when each possesses a functional groupcapable of reacting with the other. For example, a nucleophilic group,such as an amino or sulfhydryl group, may be capable of reacting with acarbonyl-containing group, such as an anhydride or an acid halide, orwith an alkyl group containing a good leaving group (e.g., a halide).Alternatively, a suitable chemical linker group may be used. A linkergroup can function as a spacer in order to avoid interference withbinding capabilities.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as a linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology. Alternatively a highaffinity PD-1 mimic polypeptide is linked to the cytotoxic or imagingmoiety by the use of a non-covalent binding pair, such asstreptavidin/biotin, or avidin/biotin. In these embodiments, one memberof the pair is covalently coupled to a high affinity PD-1 mimicpolypeptide and the other member of the binding pair is covalentlycoupled to the cytotoxic or imaging moiety. It may be desirable tocouple more than one cytotoxic and/or imaging moiety. Bypoly-derivatizing the high affinity PD-1 mimic polypeptide, severalstrategies may be simultaneously implemented, an antibody may be madeuseful as a contrasting agent for several visualization techniques, or atherapeutic antibody may be labeled for tracking by a visualizationtechnique.

A carrier may bear the agents in a variety of ways, including covalentbonding either directly or via a linker group, and non-covalentassociations. Suitable covalent-bond carriers include proteins such asalbumins, peptides, and polysaccharides such as aminodextran, each ofwhich have multiple sites for the attachment of moieties. A carrier mayalso bear an agent by non-covalent associations, such as non-covalentbonding or by encapsulation

Carriers and linkers specific for radionuclide agents includeradiohalogenated small molecules and chelating compounds. A radionuclidechelate may be formed from chelating compounds that include thosecontaining nitrogen and sulfur atoms as the donor atoms for binding themetal, or metal oxide, radionuclide.

Radiographic moieties for use as imaging moieties in the presentdisclosure include compounds and chelates with relatively large atoms,such as gold, iridium, technetium, barium, thallium, iodine, and theirisotopes. In some cases, less toxic radiographic imaging moieties, suchas iodine or iodine isotopes, can be utilized in the compositions andmethods of the disclosure. Such moieties may be conjugated to the highaffinity PD-1 mimic polypeptide through an acceptable chemical linker orchelation carrier. Positron emitting moieties for use in the presentdisclosure include ¹⁸F, which can be easily conjugated by a fluorinationreaction with the high affinity PD-1 mimic polypeptide. Examples of PETemitters include ⁶⁴Cu, ⁶⁸Ga, ⁸⁹Zr, and ¹⁸F.

PET imaging can include coupling a positron emitter to the protein.Examples of PET emitters include ⁶⁴Cu, ⁶⁸Ga, ⁸⁹Zr, and ¹⁸F. These agentscan be coupled to the protein using any convenient method, e.g., viachelating groups, e.g.,1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),1,4,7-Triazacyclononane-1,4,7-triacetic acid (NOTA), and Desferoxamine(DFO). Such chelators can be covalently and site-specifically attachedto the protein using any convenient method, e.g., by maleimide chemistryat free cysteine residues, e.g., engineered free cysteine residues suchas at cysteines R87C, N91C, and/or R122C (mutations corresponding toR87C, N91C, and/or R122C relative to the PD-1 protein fragment set forthin SEQ ID NO: 2).

Single-photon emission computed tomography (SPECT) moieties for use asimaging moieties in the present disclosure. SPECT is similar to positronemission tomography (PET) in its use of radioactive tracer material anddetection of gamma rays. In contrast with PET, however, the tracers usedin SPECT emit gamma radiation that is measured directly, whereas PETtracers emit positrons that annihilate with electrons up to a fewmillimeters away, causing two gamma photons to be emitted in oppositedirections.

Magnetic resonance contrast moieties can include chelates ofchromium(III), manganese(II), iron(II), nickel(II), copper(II),praseodymium(III), neodymium(III), samarium(III) and ytterbium(III) ion.Because of their very strong magnetic moment, the gadolinium(III),terbium(III), dysprosium(III), holmium(III), erbium(III), and iron(III)ions are also considered as contrast moieties.

Optically visible moieties for use as imaging moieties includefluorescent dyes, or visible-spectrum dyes, visible particles, and othervisible labeling moieties. Fluorescent dyes such as fluorescein,coumarin, rhodamine, bodipy Texas red, and cyanine dyes, are useful whensufficient excitation energy can be provided to the site to be inspectedvisually. Endoscopic visualization procedures may be more compatiblewith the use of such labels. Acceptable dyes include FDA-approved fooddyes and colors, which are non-toxic, although pharmaceuticallyacceptable dyes which have been approved for internal administration arepreferred.

The effective amount of an imaging conjugate composition to be given toa particular patient can depend on a variety of factors, several ofwhich will be different from patient to patient. A competent clinicianwill be able to determine an effective amount to facilitate thevisualization of, for example, a tumor. Dosage will depend on thetreatment of the tumor, route of administration, the nature of thetherapeutics, sensitivity of the tumor to the therapeutics, etc.

Utilizing ordinary skill, the competent clinician will be able tooptimize the dosage of a particular therapeutic or imaging compositionin the course of routine clinical trials.

A typical dose may be from 0.001 to 100 milligrams of conjugate perkilogram subject body weight. Relatively large doses, in the range of0.1 to 10 mg per kilogram of patient body weight may be used for imagingconjugates with a relatively non-toxic imaging moiety. The amountutilized will depend on the sensitivity of the imaging method, and therelative toxicity of the imaging moiety.

High affinity PD-1 mimic polypeptides of the present disclosure can beproduced by any suitable means known or later discovered in the field,e.g., produced from eukaryotic or prokaryotic cells, synthesized invitro, etc. Where the protein is produced by prokaryotic cells, it maybe further processed by unfolding, e.g., heat denaturation, DTTreduction, etc. and may be further refolded, using methods known in theart.

In some cases, a subject high affinity PD-1 mimic polypeptide includesone or more mutations corresponding to R87C, N91C, and/or R122C relativeto the PD-1 protein fragment set forth in SEQ ID NO: 2. In some cases, asubject high affinity PD-1 mimic polypeptide includes the amino acidsequence set forth in any of SEQ ID NOs: 3-25 and 39-46. As noted above,in some cases, the high affinity PD-1 mimic polypeptide includes adetectable label (e.g., a positron-emission tomography (PET) imaginglabel). In some cases, a subject high affinity PD-1 mimic polypeptideincludes one or more mutations corresponding to R87C, N91C, and/or R122Crelative to the PD-1 protein fragment set forth in SEQ ID NO: 2, andalso includes a detectable label (e.g., a positron-emission tomography(PET) imaging label). In some cases, a subject high affinity PD-1 mimicpolypeptide (e.g., a subject high affinity PD-1 mimic polypeptide thatincludes one or more mutations corresponding to R87C, N91C, and/or R122Crelative to the PD-1 protein fragment set forth in SEQ ID NO: 2)includes an imaging moiety for positron-emission tomography (PET)imaging (e.g., a PET emitters such as ⁶⁴Cu, ⁶⁸Ga, ⁸⁹Zr, and ¹⁸F), SPECT(e.g., a gamma ray emitter), and/or Fluorescencse imaging (e.g., afluorescent dye such as fluorescein, coumarin, rhodamine, bodipy Texasred, a cyanine dyes, and the like).

The polypeptides may be prepared by cell-free translation systems, orsynthetic in vitro synthesis, using conventional methods as known in theart. Various commercial synthetic apparatuses are available, forexample, automated synthesizers by Applied Biosystems, Inc., FosterCity, Calif., Beckman, etc. By using synthesizers, naturally occurringamino acids may be substituted with unnatural amino acids. Theparticular sequence and the manner of preparation will be determined byconvenience, economics, purity required, and the like.

The polypeptides may also be isolated and purified in accordance withconventional methods of recombinant synthesis. A lysate may be preparedof the expression host and the lysate purified using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, or otherpurification technique. For the most part, the compositions which areused will comprise at least 20% by weight of the desired product, moreusually at least about 75% by weight, preferably at least about 95% byweight, and for therapeutic purposes, usually at least about 99.5% byweight, in relation to contaminants related to the method of preparationof the product and its purification. Usually, the percentages will bebased upon total protein.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing coding sequences and appropriatetranscriptional/translational control signals. These methods include,for example, in vitro recombinant DNA techniques, synthetic techniquesand in vivo recombination/genetic recombination. Alternatively, RNAcapable of encoding the polypeptides of interest may be chemicallysynthesized. One of skill in the art can readily utilize well-knowncodon usage tables and synthetic methods to provide a suitable codingsequence for any of the polypeptides of the disclosure. The nucleicacids may be isolated and obtained in substantial purity. Usually, thenucleic acids, either as DNA or RNA, will be obtained substantially freeof other naturally-occurring nucleic acid sequences, generally being atleast about 50%, usually at least about 90% pure and are typically“recombinant,” e.g., flanked by one or more nucleotides with which it isnot normally associated on a naturally occurring chromosome. The nucleicacids of the disclosure can be provided as a linear molecule or within acircular molecule, and can be provided within autonomously replicatingmolecules (vectors) or within molecules without replication sequences.Expression of the nucleic acids can be regulated by their own or byother regulatory sequences known in the art. The nucleic acids of thedisclosure can be introduced into suitable host cells using a variety oftechniques available in the art.

According to the present disclosure, high affinity PD-1 mimicpolypeptides can be provided in pharmaceutical compositions(pharmaceutical formulations) suitable for therapeutic use, e.g., forhuman treatment. In some embodiments, pharmaceutical compositions of thepresent disclosure include one or more therapeutic entities of thepresent disclosure or pharmaceutically acceptable salts, esters orsolvates thereof. In some other embodiments, pharmaceutical compositionsof the present disclosure include one or more therapeutic entities ofthe present disclosure in combination with another therapeutic agent,e.g., another anti-tumor agent.

Therapeutic entities of the present disclosure are often administered aspharmaceutical compositions (pharmaceutical formulations) comprising anactive therapeutic agent and a other pharmaceutically acceptableexcipient. The preferred form depends on the intended mode ofadministration and therapeutic application. The compositions can alsoinclude, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like

In still some other embodiments, pharmaceutical compositions of thepresent disclosure can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized Sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes).

In some embodiments, a subject high affinity PD-1 mimic polypeptide ismultispecific (e.g., bispecific). The terms “multispecific” or“bispecific” are commonly used in the art to refer to antibodies thatrecognize two or more different antigens by virtue of possessing atleast one region (e.g., derived from a variable region of a firstantibody) that is specific for a first antigen, and at least a secondregion (e.g., derived from a variable region of a second antibody) thatis specific for a second antigen (These antibodies are also known asbifunctional antibodies or multifunctional antibodies). A bispecificantibody specifically binds to two target antigens and is thus one typeof multispecific antibody. Multispecific antibodies can be produced byrecombinant DNA methods or include, but are not limited to, antibodiesproduced chemically by any convenient method. Bispecific antibodiesinclude all antibodies or conjugates of antibodies, or polymeric formsof antibodies which are capable of recognizing two different antigens.Bispecific antibodies include antibodies that have been reduced andreformed so as to retain their bivalent characteristics and toantibodies that have been chemically coupled so that they can haveseveral antigen recognition sites for each antigen.

In some embodiments, a subject high affinity PD-1 mimic polypeptide ismultispecific (e.g., bispecific). For example, a subject high affinityPD-1 mimic polypeptide can be multispecific (e.g., bispecific) such thata first region of the polypeptide corresponds to a subject high affinityPD-1 mimic polypeptide sequence (which specifically binds PD-L1), and asecond region (the fusion partner) (e.g., an antibody derived sequence,e.g., a binding region of an antibody comprising that CDRs of theantibody; a specific binding polypeptide; a binding portion of a ligand;a binding portion of a receptor, etc.) that specifically binds toanother target (e.g., antigen, a receptor, a ligand, etc.). For example,in some cases, a high affinity PD-1 mimic polypeptide is fused to asecond polypeptide (a fusion partner) that binds specifically to atarget sequence other than PD-L1. In some cases, a high affinity PD-1mimic polypeptide is fused to a second polypeptide (a fusion partner)that binds specifically to a target other than PD-L1 (thus, such amultimeric high affinity PD-1 mimic polypeptide can be bispecific, andwould therefore bind 2 different targets/moieties).

In some cases, a subject multimeric high affinity PD-1 mimic polypeptidealters signaling as a result of the fusion partner binding to itstarget. For example, in some cases, the fusion partner includes, as afusion partner, a ligand or a binding region of a ligand (e.g., acytokine, an attenuated cytokine, etc.), and the multimeric highaffinity PD-1 mimic polypeptide alters signaling when the ligand bindsto its target (e.g., a receptor). Likewise, in some cases, the fusionpartner includes, as a fusion partner, a receptor or a binding region ofa receptor, and the multimeric high affinity PD-1 mimic polypeptidealters signaling when the receptor binds to its target (e.g., a ligand).

Examples of suitable fusion partners include, but are not limited to thebinding sequences from antibodies against cancer cell markers such asCD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD52, CD56, CD70,CD96, CD97, CD99, CD123, CD279 (PD-1), EGFR, HER2, CD117, C-Met, PTHR2,HAVCR2 (TIM3), etc. Examples of antibodies with CDRs that providespecific binding to a cancer cell marker include, but are not limitedto: CETUXIMAB (binds EGFR), PANITUMUMAB (binds EGFR), RITUXIMAB (bindsCD20), TRASTUZUMAB (binds HER2), PERTUZUMAB (binds HER2), ALEMTUZUMAB(binds CD52), BRENTUXIMAB (binds CD30), and the like.

Examples of suitable fusion partners include a cytokine; an attenuatedcytokine; a 41 BB-agonist; CD40-agonist; an inhibitor of BTLA and/orCD160; and an inhibitor of TIM3 and/or CEACAM1. For example, in somecases, a high affinity PD-1 mimic polypeptide is a multispecific highaffinity PD-1 mimic polypeptide, and is fused to one or more fusionpartners selected from: a cytokine; an attenuated cytokine; a 41BB-agonist; CD40-agonist; an inhibitor of BTLA and/or CD160; and aninhibitor of TIM3 and/or CEACAM1. For example, a multispecific highaffinity PD-1 mimic polypeptide can be fused to a fusion partner that isa modified cytokine that has a decreased affinity (reduced relative to acorresponding wild type cytokine) for its ligand/receptor. Such amodified cytokine is referred to herein as an ‘attenuated cytokine’. Anexample of an attenuated cytokine (one example of a possible fusionpartner) is an IL-2 protein that has mutations that decrease affinityfor two of the IL-2 receptor subunits (e.g., F42A to decrease binding toCD25 and D20T to decrease binding to CD122) (e.g., see SEQ ID NO: 39).

In some cases a high affinity PD-1 mimic polypeptide is a multispecifichigh affinity PD-1 mimic polypeptide, and has a fusion partner that is a41 BB-agonist (e.g., 41 BBL, e.g., see SEQ ID NO: 40). In some cases ahigh affinity PD-1 mimic polypeptide is a multispecific high affinityPD-1 mimic polypeptide, and has a fusion partner that is a CD40-agonist(e.g., CD40L, e.g., see SEQ ID NO: 41). In some cases a high affinityPD-1 mimic polypeptide is a multispecific high affinity PD-1 mimicpolypeptide, and has a fusion partner that is an inhibitor of BTLAand/or CD160 (e.g., see SEQ ID NO: 42). In some cases a high affinityPD-1 mimic polypeptide is a multispecific high affinity PD-1 mimicpolypeptide, and has a fusion partner that is an inhibitor of TIM3and/or CEACAM1 (e.g., see SEQ ID NO: 43).

In some embodiments, a subject high affinity PD-1 mimic polypeptide anda fusion partner are separated by a linker (e.g., a linker polypeptide).A linker polypeptide may have any of a variety of amino acid sequences.Proteins can be joined by a linker polypeptide can be of a flexiblenature (e.g., a flexible linker polypeptide), although other chemicallinkages are not excluded. Suitable linkers include polypeptides ofbetween about 6 amino acids and about 40 amino acids in length, orbetween about 6 amino acids and about 25 amino acids in length. Theselinkers can be produced by using synthetic, linker-encodingoligonucleotides to couple the proteins. Peptide linkers with a degreeof flexibility can be used. The linking peptides may have virtually anyamino acid sequence, bearing in mind that the in some case, linkers willhave a sequence that results in a generally flexible peptide. The use ofsmall amino acids, such as glycine and alanine, are of use in creating aflexible peptide. The creation of such sequences is routine to those ofskill in the art. A variety of different linkers are commerciallyavailable and are considered suitable for use.

Examples of linker polypeptides include glycine polymers (G)_(n),glycine-serine polymers (including, for example, (GS)_(n), GSGGS_(n)(SEQ ID NO: 47), GGSGGS_(n) (SEQ ID NO: 48), and GGGS_(n) (SEQ ID NO:49), where n is an integer of at least one (e.g., where n is an integerof one, two, three, four, five, six, seven, eight, nine, ten, or greaterthan ten), glycine-alanine polymers, alanine-serine polymers. Exemplarylinkers can comprise amino acid sequences including, but not limited to,GGSG (SEQ ID NO: 50), GGSGG (SEQ ID NO: 51), GSGSG (SEQ ID NO: 52),GSGGG (SEQ ID NO: 53), GGGSG (SEQ ID NO: 54), GSSSG (SEQ ID NO: 55), andthe like. The ordinarily skilled artisan will recognize that design of apeptide conjugated to any elements described above can include linkersthat are all or partially flexible, such that the linker can include aflexible linker as well as one or more portions that confer lessflexible structure.

Nucleic Acids.

The disclosure also provides isolated nucleic acids encoding a subjecthigh affinity PD-1 mimic polypeptide, vectors and host cells comprisingthe nucleic acid, and recombinant techniques for the production of thehigh affinity PD-1 mimic polypeptide. For recombinant production of thehigh affinity PD-1 mimic polypeptide, the nucleic acid encoding the highaffinity PD-1 mimic polypeptide can be inserted into a replicable vectorfor further cloning (amplification of the DNA) or for expression. DNAencoding a subject high affinity PD-1 mimic polypeptide can be readilyisolated and sequenced using conventional procedures. Many vectors areavailable. The vector components generally include, but are not limitedto, one or more of the following: a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence.

A subject high affinity PD-1 mimic polypeptide of this disclosure may beproduced recombinantly not only directly, but also as a fusionpolypeptide with a heterologous or homologous polypeptide, which caninclude a signal sequence or other polypeptide having a specificcleavage site at the N-terminus of the mature protein or polypeptide, animmunoglobulin constant region sequence, and the like. A heterologoussignal sequence selected preferably may be one that is recognized andprocessed (i.e., cleaved by a signal peptidase) by the host cell. Forprokaryotic host cells that do not recognize and process the nativeantibody signal sequence, the signal sequence is substituted by aprokaryotic signal sequence selected.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated prior to isolation. Anisolated nucleic acid molecule is other than in the form or setting inwhich it can be found in nature. Isolated nucleic acid moleculestherefore are distinguished from the nucleic acid molecule as it existsin natural cells.

Examples of suitable host cells for cloning or expressing subjectnucleic acids include, but are not necessary limited to prokaryote,yeast, or higher eukaryote cells. Examples of useful mammalian host celllines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol. 36:59(1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1.982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2). Host cells are transformed with the above-described expressionor cloning vectors for high affinity PD-1 mimic polypeptide productionand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Introduction of Nucleic Acids.

In some cases, as subject high affinity PD-1 mimic polypeptide isadministered to an individual by providing the high affinity PD-1 mimicpolypeptide as a nucleic acid (e.g., an RNA, e.g., an mRNA; or a DNA,e.g., a recombinant expression vector, a linear DNA, a circular DNA, aplasmid, a viral vector, etc.) encoding the high affinity PD-1 mimicpolypeptide. This disclosure provides such methods and also the nucleicacids for such methods.

For example, an mRNA encoding a subject high affinity PD-1 mimicpolypeptide can be introduced into a cell, and the cell can then secretthe translated protein. As another example, a DNA (e.g., a recombinantexpression vector, a linear DNA, a circular DNA, a plasmid, a viralvector, etc.) encoding a subject high affinity PD-1 mimic polypeptidecan be introduced into a cell and the cell can then produce and secretthe encoded protein. Therefore, in some cases, a nucleic acid encoding asubject high affinity PD-1 mimic polypeptide includes a nucleotidesequence encoding a signal sequence (e.g., upstream of and in frame withthe nucleotide sequence that encodes the high affinity PD-1 mimicpolypeptide). As would be readily recognized by one of ordinary skill inthe art, a signal sequence as referred to here is an amino acid sequenceat or near the amino terminus of a nascent protein that can berecognized by the signal recognition particle of a eukaryotic cell,resulting in transport of the protein into the secretory pathway of thecell, thus facilitating secretion of a protein from the cell (e.g., thesignal sequence can be cleaved from the protein). Any convenient signalsequence can be used.

In some cases, a nucleic acid encoding a subject high affinity PD-1mimic polypeptide is introduced into a cell (e.g., in vivo, ex vivo, invitro) and the cell can then produce and secret the encoded protein. Insome cases, the cell is in vitro. In some cases, the cell is ex vivo. Insome cases, the cell is in vivo. For example, in some cases, a nucleicacid encoding a high affinity PD-1 mimic polypeptide is introduced intoa cell that is in vivo (e.g., in some cases, a nucleic acid encoding ahigh affinity PD-1 mimic polypeptide is introduced into a cell in vivoby administering the nucleic acid to an individual). In some cases, anucleic acid encoding a subject high affinity PD-1 mimic polypeptide isintroduced into a cell (e.g., ex vivo, in vitro) and the cell is thenintroduced into an individual. In some cases, the cell is autologous tothe individual (e.g., the cell was isolated from the individual or isthe progeny of a cell that was isolated from the individual).

In some cases (e.g., in any of the above scenarios, e.g., in vitro, exvivo, in vivo), the cell into which a nucleic acid encoding a subjecthigh affinity PD-1 mimic polypeptide is introduced is an immune cell(e.g., a leukocyte, a T cell, a CD8 T cell, a CD4 T cell, amemory/effector T cell, a B cell, an antigen presenting cell (APC), adendritic cell, a macrophage, a monocyte, an NK cell, and the like). Insome cases (e.g., in any of the above scenarios, e.g., in vitro, exvivo, in vivo), the cell into which a nucleic acid encoding a subjecthigh affinity PD-1 mimic polypeptide is introduced is a stem cell (e.g.,a hematopoietic stem cell, a pluripotent stem cell, a multipotent stemcell, a tissue restricted stem cell, etc.). In some cases (e.g., in anyof the above scenarios, e.g., in vitro, ex vivo, in vivo), the cell intowhich a nucleic acid encoding a subject high affinity PD-1 mimicpolypeptide is introduced is an immune cell (e.g., a leukocyte, a Tcell, a CD8 T cell, a CD4 T cell, a memory/effector T cell, a B cell, anantigen presenting cell (APC), a dendritic cell, a macrophage, amonocyte, an NK cell, and the like) or a stem cell (e.g., ahematopoietic stem cell, a pluripotent stem cell, a multipotent stemcell, a tissue restricted stem cell, etc.).

In some cases (e.g., in any of the above scenarios, e.g., in vitro, exvivo, in vivo), the cell into which a nucleic acid encoding a subjecthigh affinity PD-1 mimic polypeptide is introduced is a T cell with anengineered T cell receptor (TCR) (such a cell is also referred to hereinas a “TCR-engineered T cell”). As used herein the term “TCR-engineered Tcell” refers to any T-cell having a T cell receptor that is heterologousto the T cell. Suitable examples include, but are not limited to (i) a Tcell that includes a chimeric antigen receptor (CAR) (such a cell isalso referred to herein as a “CAR-T cell” or an “engineered CAR-Tcell”); and (ii) a T cell that includes a heterologous TCR that binds toan antigen such as a cancer antigen, e.g., MART1, NY-ESO-1, p53, and thelike (e.g., such a cell can include a nucleic acid encoding theTcR-alpha and TcR-beta polypeptides of a heterologous TCR, such as a TCRthat binds to an antigen such as a cancer antigen, e.g., MART1,NY-ESO-1, p53, and the like).

In some cases, a suitable TCR-engineered T cell can have an engineeredTCR (e.g., a heterologous TCR that binds to an antigen, a CAR, etc.)that binds to a cancer marker (e.g., CD19, CD20, CD22, CD24, CD25, CD30,CD33, CD38, CD44, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279(PD-1), EGFR, HER2, CD117, C-Met, PTHR2, and/or HAVCR2 (TIM3)). In somecases, a suitable TCR-engineered T cell can have an engineered TCR(e.g., a heterologous TCR that binds to an antigen, a CAR, etc.) thatbinds to a target antigen selected from PD-L1 (e.g., a CAR derived froman anti-PD-L1 antibody) and PD-1 (e.g., a CAR derived from an anti-PD-1antibody). In some cases, a suitable TCR-engineered T cell can have anengineered TCR (e.g., a heterologous TCR that binds to an antigen, aCAR, etc.) that binds to PD-L1 (e.g., a CAR derived from an anti-PD-L1antibody). In some cases, a suitable TCR-engineered T cell can have anengineered TCR (e.g., a heterologous TCR that binds to an antigen, aCAR, etc.) that binds to PD-1 (e.g., a CAR derived from an anti-PD-1antibody). In some cases, a suitable TCR-engineered T cell can have anengineered TCR (e.g., a heterologous TCR that binds to an antigen, aCAR, etc.) that binds to CD19 (e.g., the 1 D3 CAR).

In some cases, a nucleic acid encoding a subject high affinity PD-1mimic polypeptide also includes a nucleotide sequence encoding a T cellreceptor (TCR). In some such case, the nucleic acid includes nucleotidesequences encoding both a TCR alpha polypeptide, and a TCR betapolypeptide of the TCR. In some cases, a nucleic acid encoding a subjecthigh affinity PD-1 mimic polypeptide also includes a nucleotide sequenceencoding the TcR-alpha and TcR-beta polypeptides of a heterologous TCR(such as a TCR that binds to an antigen such as a cancer antigen, e.g.,MART1, NY-ESO-1, p53, and the like) and/or encodes a CAR (e.g., a firstgeneration CAR, a second generation CAR, a third generation CAR, and thelike). The various components, including the sequence encoding a subjecthigh affinity PD-1 mimic polypeptide as well as the sequences encodingthe TcR-alpha and TcR-beta polypeptides of a heterologous TCR and thesequence encoding a CAR are modular. Thus, the various sequences caneach be operably linked to the same or different promoters. Thosecomponents that are operably linked to a same promoter can be separatedby sequences that allow for the proteins to eventually exist as separatepolypeptides (e.g., an internal ribosome entry site (IRES), 2A peptidesequences, etc.). Examples of various possible arrangements include, butare not limited to those depicted in FIGS. 20A-20E and FIGS. 21A-21D.Thus, examples of nucleic acids encoding a subject high affinity PD-1mimic polypeptide include, but are not limited to, those depicted inFIGS. 20A-20E and FIGS. 21A-21D.

A “vector” or “expression vector” is a replicon, such as plasmid, phage,virus, or cosmid, to which another DNA segment, i.e. an “insert”, may beattached so as to bring about the replication of the attached segment ina cell.

An “expression cassette” comprises a DNA coding sequence operably linkedto a promoter. “Operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. For instance, a promoter is operablylinked to a coding sequence if the promoter affects its transcription orexpression.

The terms “recombinant expression vector,” or “DNA construct” or“expression vector” and similar terms of the art are usedinterchangeably herein to refer to a DNA molecule comprising a vectorand at least one insert. Recombinant expression vectors can be generatedfor the purpose of expressing and/or propagating the insert(s), or forthe construction of other recombinant nucleotide sequences. Theinsert(s) (e.g., a nucleotide sequence encoding a subject high affinityPD-1 mimic polypeptide) may or may not be operably linked to a promotersequence and may or may not be operably linked to DNA regulatorysequences. Thus in some cases, a nucleotide sequence encoding a subjecthigh affinity PD-1 mimic polypeptide is operably linked to a promoter(e.g., one that is operable in a desired cell type, e.g., a eukaryoticcell, a mammalian cell, a primate cell, a human cell, an immune cell, aleukocyte, a T cell, a CD8 T cell, a CD4 T cell, a memory/effector Tcell, a B cell, an antigen presenting cell (APC), a dendritic cell, amacrophage, a monocyte, an NK cell, a stem cell, a hematopoietic stemcell, a pluripotent stem cell, a multipotent stem cell, a tissuerestricted stem cell, etc.).

A promoter can be a constitutively active promoter (i.e., a promoterthat is constitutively in an active/“ON” state), it may be an induciblepromoter (i.e., a promoter whose state, active/“ON” or inactive/“OFF”,is controlled by an external stimulus, e.g., the presence of aparticular temperature, compound, or protein.), it may be a spatiallyrestricted promoter (i.e., transcriptional control element, enhancer,etc.)(e.g., tissue specific promoter, cell type specific promoter,etc.), and it may be a temporally restricted promoter (i.e., thepromoter is in the “ON” state or “OFF” state during specific stages ofembryonic development or during specific stages of a biological process,e.g., hair follicle cycle in mice).

Suitable promoters can be derived from viruses and can therefore bereferred to as viral promoters, or they can be derived from anyorganism, including prokaryotic or eukaryotic organisms. Suitablepromoters can be used to drive expression by any RNA polymerase (e.g.,pol I, pol II, pol III). Exemplary promoters include, but are notlimited to the SV40 early promoter, mouse mammary tumor virus longterminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP);a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promotersuch as the CMV immediate early promoter region (CMVIE), a rous sarcomavirus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishiet al., Nature Biotechnology 20, 497-500 (2002)), an enhanced U6promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31(17)), ahuman H1 promoter (H1), and the like.

Examples of inducible promoters include, but are not limited to T7 RNApolymerase promoter, T3 RNA polymerase promoter,Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter,lactose induced promoter, heat shock promoter, Tetracycline-regulatedpromoter, Steroid-regulated promoter, Metal-regulated promoter, estrogenreceptor-regulated promoter, etc. Inducible promoters can therefore beregulated by molecules including, but not limited to, doxycycline; RNApolymerase, e.g., T7 RNA polymerase; an estrogen receptor; an estrogenreceptor fusion; etc.

In some embodiments, the promoter is a spatially restricted promoter(i.e., cell type specific promoter, tissue specific promoter, etc.) suchthat in a multi-cellular organism, the promoter is active (i.e., “ON”)in a subset of specific cells. Spatially restricted promoters may alsobe referred to as enhancers, transcriptional control elements, controlsequences, etc. Any convenient spatially restricted promoter may be usedand the choice of suitable promoter (e.g., a brain specific promoter, apromoter that drives expression in a subset of neurons, a promoter thatdrives expression in the germline, a promoter that drives expression inthe lungs, a promoter that drives expression in muscles, a promoter thatdrives expression in islet cells of the pancreas, etc.) will depend onthe organism. For example, various spatially restricted promoters areknown for plants, flies, worms, mammals, mice, etc. Thus, a spatiallyrestricted promoter can be used to regulate the expression of a nucleicacid encoding a subject site-directed modifying polypeptide in a widevariety of different tissues and cell types, depending on the organism.Some spatially restricted promoters are also temporally restricted suchthat the promoter is in the “ON” state or “OFF” state during specificstages of embryonic development or during specific stages of abiological process (e.g., hair follicle cycle in mice).

For illustration purposes, examples of spatially restricted promotersinclude, but are not limited to, neuron-specific promoters,adipocyte-specific promoters, cardiomyocyte-specific promoters, smoothmuscle-specific promoters, photoreceptor-specific promoters, etc.Neuron-specific spatially restricted promoters include, but are notlimited to, a neuron-specific enolase (NSE) promoter (see, e.g., EMBLHSENO2, X51956); an aromatic amino acid decarboxylase (AADC) promoter; aneurofilament promoter (see, e.g., GenBank HUMNFL, L04147); a synapsinpromoter (see, e.g., GenBank HUMSYNIB, M55301); a thy-1 promoter (see,e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn, et al. (2010) Nat.Med. 16(10):1161-1166); a serotonin receptor promoter (see, e.g.,GenBank S62283); a tyrosine hydroxylase promoter (TH) (see, e.g., Oh etal. (2009) Gene Ther 16:437; Sasaoka et al. (1992) Mol. Brain Res.16:274; Boundy et al. (1998) J. Neurosci. 18:9989; and Kaneda et al.(1991) Neuron 6:583-594); a GnRH promoter (see, e.g., Radovick et al.(1991) Proc. Natl. Acad. Sci. USA 88:3402-3406); an L7 promoter (see,e.g., Oberdick et al. (1990) Science 248:223-226); a DNMT promoter (see,e.g., Bartge et al. (1988) Proc. Natl. Acad. Sci. USA 85:3648-3652); anenkephalin promoter (see, e.g., Comb et al. (1988) EMBO J.17:3793-3805); a myelin basic protein (MBP) promoter; aCa2+-calmodulin-dependent protein kinase II-alpha (CamKIIα) promoter(see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93:13250;and Casanova et al. (2001) Genesis 31:37); a CMVenhancer/platelet-derived growth factor-β promoter (see, e.g., Liu etal. (2004) Gene Therapy 11:52-60); and the like.

Adipocyte-specific spatially restricted promoters include, but are notlimited to aP2 gene promoter/enhancer, e.g., a region from −5.4 kb to+21 bp of a human aP2 gene (see, e.g., Tozzo et al. (1997) Endocrinol.138:1604; Ross et al. (1990) Proc. Natl. Acad. Sci. USA 87:9590; andPavjani et al. (2005) Nat. Med. 11:797); a glucose transporter-4 (GLUT4)promoter (see, e.g., Knight et al. (2003) Proc. Natl. Acad. Sci. USA100:14725); a fatty acid translocase (FAT/CD36) promoter (see, e.g.,Kuriki et al. (2002) Biol. Pharm. Bull. 25:1476; and Sato et al. (2002)J. Biol. Chem. 277:15703); a stearoyl-CoA desaturase-1 (SCD1) promoter(Tabor et al. (1999) J. Biol. Chem. 274:20603); a leptin promoter (see,e.g., Mason et al. (1998) Endocrinol. 139:1013; and Chen et al. (1999)Biochem. Biophys. Res. Comm. 262:187); an adiponectin promoter (see,e.g., Kita et al. (2005) Biochem. Biophys. Res. Comm. 331:484; andChakrabarti (2010) Endocrinol. 151:2408); an adipsin promoter (see,e.g., Platt et al. (1989) Proc. Natl. Acad. Sci. USA 86:7490); aresistin promoter (see, e.g., Seo et al. (2003) Molec. Endocrinol.17:1522); and the like.

Cardiomyocyte-specific spatially restricted promoters include, but arenot limited to control sequences derived from the following genes:myosin light chain-2, α-myosin heavy chain, AE3, cardiac troponin C,cardiac actin, and the like. Franz et al. (1997) Cardiovasc. Res.35:560-566; Robbins et al. (1995) Ann. N.Y. Acad. Sci. 752:492-505; Linnet al. (1995) Circ. Res. 76:584-591; Parmacek et al. (1994) Mol. Cell.Biol. 14:1870-1885; Hunter et al. (1993) Hypertension 22:608-617; andSartorelli et al. (1992) Proc. Natl. Acad. Sci. USA 89:4047-4051.

Smooth muscle-specific spatially restricted promoters include, but arenot limited to an SM22α promoter (see, e.g., Akyürek et al. (2000) Mol.Med. 6:983; and U.S. Pat. No. 7,169,874); a smoothelin promoter (see,e.g., WO 2001/018048); an α-smooth muscle actin promoter; and the like.For example, a 0.4 kb region of the SM22α promoter, within which lie twoCArG elements, has been shown to mediate vascular smooth musclecell-specific expression (see, e.g., Kim, et al. (1997) Mol. Cell. Biol.17, 2266-2278; Li, et al., (1996) J. Cell Biol. 132, 849-859; andMoessler, et al. (1996) Development 122, 2415-2425).

Photoreceptor-specific spatially restricted promoters include, but arenot limited to, a rhodopsin promoter; a rhodopsin kinase promoter (Younget al. (2003) Ophthalmol. Vis. Sci. 44:4076); a beta phosphodiesterasegene promoter (Nicoud et al. (2007) J. Gene Med. 9:1015); a retinitispigmentosa gene promoter (Nicoud et al. (2007) supra); aninterphotoreceptor retinoid-binding protein (IRBP) gene enhancer (Nicoudet al. (2007) supra); an IRBP gene promoter (Yokoyama et al. (1992) ExpEye Res. 55:225); and the like.

The terms “DNA regulatory sequences,” “control elements,” and“regulatory elements,” used interchangeably herein, refer totranscriptional and translational control sequences, such as promoters,enhancers, polyadenylation signals, terminators, protein degradationsignals, and the like, that provide for and/or regulate transcription ofa non-coding sequence (e.g., DNA-targeting RNA) or a coding sequence(e.g., site-directed modifying polypeptide, or Cas9/Csn1 polypeptide)and/or regulate translation of an encoded polypeptide.

Suitable expression vectors include, but are not limited to, viralvectors (e.g., viral vectors based on vaccinia virus; poliovirus;adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549,1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al.,Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali etal., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulskiet al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988)166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40;herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshiet al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816,1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosisvirus, and vectors derived from retroviruses such as Rous Sarcoma Virus,Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, humanimmunodeficiency virus, myeloproliferative sarcoma virus, and mammarytumor virus); and the like.

Numerous suitable expression vectors are known to those of skill in theart, and many are commercially available. The following vectors areprovided by way of example; for eukaryotic host cells: pXT1, pSG5(Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, anyother vector may be used so long as it is compatible with the host cell.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation control elements, includingconstitutive and inducible promoters, transcription enhancer elements,transcription terminators, etc. may be used in the expression vector(see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

Also provided in this disclosure are cells that include a nucleic acid(e.g., as described above) that includes a nucleotide sequence encodinga subject high affinity PD-1 mimic polypeptide. Such a cell can be acell from any organism (e.g., a bacterial cell, an archaeal cell, a cellof a single-cell eukaryotic organism, a plant cell, an algal cell, afungal cell (e.g., a yeast cell), an animal cell, a cell from aninvertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode,etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile,bird, mammal), a cell from a mammal, a cell from a rodent, a cell from ahuman, etc.).

Methods of Use

Methods are provided for treating, reducing and/or or preventing cancer;treating, reducing and/or or preventing infection (e.g., chronicinfection); and/or for treating, reducing and/or or preventing animmunological disease or disorder (e.g., an inflammatory disease, acondition associated with immunosuppression, etc.)(e.g., multiplesclerosis, arthritis, and the like). For example, in some cases, asubject high affinity PD-1 mimic polypeptide can be used as an immunestimulant (e.g., used for immunopotentiation).

In some cases, subject methods result in the reduction in the number ofcancer cells in an individual. In some cases, subject methods result ina reduction of tumor size. In some cases, subject methods result in areduction of tumor size. In some cases, subject methods reduce thebinding of PD-1 on a first cell with PD-L1 on a second cell.

In some cases, a subject method is a method of diagnosing and/orprognosing for an individual (e.g., diagnosing and/or prognosing cancerin an individual). For example, high affinity PD-1 mimic polypeptidesare useful as imaging agents, e.g., when conjugated to a detectablelabel such as a PET label and/or fluorescent label (e.g., as describedelsewhere in this disclosure), which can be used for various purposessuch as diagnostic/prognostic reagents. For example, in some cases asubject method is a method of diagnosing or prognosing cancer in anindividual (e.g., a cancer in which the presence, level, and/or locationof detectable PD-L1 can be diagnostic and/or prognostic).

Inhibition of PD-1 mediated cellular signaling by therapeutic agents(e.g., a subject high-affinity PD-1 mimic polypeptide) can activateimmune cells (e.g., T cells, B cells, NK cells, etc.), and thereforeenhance immune cell functions such as inhibiting cancer cell growthand/or viral infection, and restore immune surveillance and immunememory function to treat human disease. Examples of symptoms, illnesses,and/or diseases that can be treated with a subject high-affinity PD-1mimic polypeptide include, but are not limited to cancer (any form ofcancer, including but not limited to: carcinomas, soft tissue tumors,sarcomas, teratomas, melanomas, leukemias, lymphomas, brain cancers,solid tumors, mesothelioma (MSTO), etc.); infection (e.g., chronicinfection); and/or an immunological disease or disorder (e.g., aninflammatory disease)(e.g., multiple sclerosis, arthritis, and thelike). For example, in some cases, a subject high affinity PD-1 mimicpolypeptide can be used as an immune stimulant (e.g., used forimmunopotentiation). Any disease, disorder or ailment that involvesimmunosuppression (e.g., caused by an immunosuppressive signal inducedby PD-1, PD-L1, or PD-L2 signaling) can be treated using a subject highaffinity PD-1 mimic polypeptide.

Any cancer is a suitable cancer to be treated by the subject methods andcompositions. In some cases, cancer cells of the cancer express PD-L1(i.e. are positive for PD-L1 expression). In some cases, cancer cells ofthe cancer do not express PD-L1, however such cancers can still betreated with a subject high-affinity PD-1 mimic polypeptide (e.g., dueto immunopotentiation by the high-affinity PD-1 mimic polypeptide).

As used herein “cancer” includes any form of cancer, including but notlimited to solid tumor cancers (e.g., lung, prostate, breast, bladder,colon, ovarian, pancreas, kidney, liver, glioblastoma, medulloblastoma,leiomyosarcoma, head & neck squamous cell carcinomas, melanomas,neuroendocrine; etc.) and liquid cancers (e.g., hematological cancers);carcinomas; soft tissue tumors; sarcomas; teratomas; melanomas;leukemias; lymphomas; and brain cancers, including minimal residualdisease, and including both primary and metastatic tumors. Any cancer isa suitable cancer to be treated by the subject methods and compositions.In some cases, the cancer cells express PD-L1. In some cases, the cancercells do not express PD-L1 (e.g., in such cases, cells of the immunesystem of the individual being treated express PD-L1).

Carcinomas are malignancies that originate in the epithelial tissues.Epithelial cells cover the external surface of the body, line theinternal cavities, and form the lining of glandular tissues. Examples ofcarcinomas include, but are not limited to: adenocarcinoma (cancer thatbegins in glandular (secretory) cells), e.g., cancers of the breast,pancreas, lung, prostate, and colon can be adenocarcinomas;adrenocortical carcinoma; hepatocellular carcinoma; renal cellcarcinoma; ovarian carcinoma; carcinoma in situ; ductal carcinoma;carcinoma of the breast; basal cell carcinoma; squamous cell carcinoma;transitional cell carcinoma; colon carcinoma; nasopharyngeal carcinoma;multilocular cystic renal cell carcinoma; oat cell carcinoma; large celllung carcinoma; small cell lung carcinoma; non-small cell lungcarcinoma; and the like. Carcinomas may be found in prostrate, pancreas,colon, brain (usually as secondary metastases), lung, breast, skin, etc.

Soft tissue tumors are a highly diverse group of rare tumors that arederived from connective tissue. Examples of soft tissue tumors include,but are not limited to: alveolar soft part sarcoma; angiomatoid fibroushistiocytoma; chondromyoxid fibroma; skeletal chondrosarcoma;extraskeletal myxoid chondrosarcoma; clear cell sarcoma; desmoplasticsmall round-cell tumor; dermatofibrosarcoma protuberans; endometrialstromal tumor; Ewing's sarcoma; fibromatosis (Desmoid); fibrosarcoma,infantile; gastrointestinal stromal tumor; bone giant cell tumor;tenosynovial giant cell tumor; inflammatory myofibroblastic tumor;uterine leiomyoma; leiomyosarcoma; lipoblastoma; typical lipoma; spindlecell or pleomorphic lipoma; atypical lipoma; chondroid lipoma;well-differentiated liposarcoma; myxoid/round cell liposarcoma;pleomorphic liposarcoma; myxoid malignant fibrous histiocytoma;high-grade malignant fibrous histiocytoma; myxofibrosarcoma; malignantperipheral nerve sheath tumor; mesothelioma; neuroblastoma;osteochondroma; osteosarcoma; primitive neuroectodermal tumor; alveolarrhabdomyosarcoma; embryonal rhabdomyosarcoma; benign or malignantschwannoma; synovial sarcoma; Evan's tumor; nodular fasciitis;desmoid-type fibromatosis; solitary fibrous tumor; dermatofibrosarcomaprotuberans (DFSP); angiosarcoma; epithelioid hemangioendothelioma;tenosynovial giant cell tumor (TGCT); pigmented villonodular synovitis(PVNS); fibrous dysplasia; myxofibrosarcoma; fibrosarcoma; synovialsarcoma; malignant peripheral nerve sheath tumor; neurofibroma; andpleomorphic adenoma of soft tissue; and neoplasias derived fromfibroblasts, myofibroblasts, histiocytes, vascular cells/endothelialcells and nerve sheath cells.

A sarcoma is a rare type of cancer that arises in cells of mesenchymalorigin, e.g., in bone or in the soft tissues of the body, includingcartilage, fat, muscle, blood vessels, fibrous tissue, or otherconnective or supportive tissue. Different types of sarcoma are based onwhere the cancer forms. For example, osteosarcoma forms in bone,liposarcoma forms in fat, and rhabdomyosarcoma forms in muscle. Examplesof sarcomas include, but are not limited to:

askin's tumor; sarcoma botryoides; chondrosarcoma; ewing's sarcoma;malignant hemangioendothelioma; malignant schwannoma; osteosarcoma; andsoft tissue sarcomas (e.g., alveolar soft part sarcoma; angiosarcoma;cystosarcoma phyllodesdermatofibrosarcoma protuberans (DFSP); desmoidtumor; desmoplastic small round cell tumor; epithelioid sarcoma;extraskeletal chondrosarcoma; extraskeletal osteosarcoma; fibrosarcoma;gastrointestinal stromal tumor (GIST); hemangiopericytoma;hemangiosarcoma (more commonly referred to as “angiosarcoma”); kaposi'ssarcoma; leiomyosarcoma; liposarcoma; lymphangiosarcoma; malignantperipheral nerve sheath tumor (MPNST); neurofibrosarcoma; synovialsarcoma; undifferentiated pleomorphic sarcoma, and the like).

A teratoma is a type of germ cell tumor that may contain severaldifferent types of tissue (e.g., can include tissues derived from anyand/or all of the three germ layers: endoderm, mesoderm, and ectoderm),including for example, hair, muscle, and bone. Teratomas occur mostoften in the ovaries in women, the testicles in men, and the tailbone inchildren.

Melanoma is a form of cancer that begins in melanocytes (cells that makethe pigment melanin). It may begin in a mole (skin melanoma), but canalso begin in other pigmented tissues, such as in the eye or in theintestines.

Leukemias are cancers that start in blood-forming tissue, such as thebone marrow, and causes large numbers of abnormal blood cells to beproduced and enter the bloodstream. For example, leukemias can originatein bone marrow-derived cells that normally mature in the bloodstream.Leukemias are named for how quickly the disease develops and progresses(e.g., acute versus chronic) and for the type of white blood cell thatis effected (e.g., myeloid versus lymphoid). Myeloid leukemias are alsocalled myelogenous or myeloblastic leukemias. Lymphoid leukemias arealso called lymphoblastic or lymphocytic leukemia. Lymphoid leukemiacells may collect in the lymph nodes, which can become swollen. Examplesof leukemias include, but are not limited to: Acute myeloid leukemia(AML), Acute lymphoblastic leukemia (ALL), Chronic myeloid leukemia(CML), and Chronic lymphocytic leukemia (CLL).

Lymphomas are cancers that begin in cells of the immune system. Forexample, lymphomas can originate in bone marrow-derived cells thatnormally mature in the lymphatic system. There are two basic categoriesof lymphomas. One kind is Hodgkin lymphoma (HL), which is marked by thepresence of a type of cell called the Reed-Sternberg cell. There arecurrently 6 recognized types of HL. Examples of Hodgkin lymphomasinclude: nodular sclerosis classical Hodgkin lymphoma (CHL), mixedcellularity CHL, lymphocyte-depletion CHL, lymphocyte-rich CHL, andnodular lymphocyte predominant HL.

The other category of lymphoma is non-Hodgkin lymphomas (NHL), whichincludes a large, diverse group of cancers of immune system cells.Non-Hodgkin lymphomas can be further divided into cancers that have anindolent (slow-growing) course and those that have an aggressive(fast-growing) course. There are currently 61 recognized types of NHL.Examples of non-Hodgkin lymphomas include, but are not limited to:AIDS-related Lymphomas, anaplastic large-cell lymphoma,angioimmunoblastic lymphoma, blastic NK-cell lymphoma, Burkitt'slymphoma, Burkitt-like lymphoma (small non-cleaved cell lymphoma),chronic lymphocytic leukemia/small lymphocytic lymphoma, cutaneousT-Cell lymphoma, diffuse large B-Cell lymphoma, enteropathy-type T-Celllymphoma, follicular lymphoma, hepatosplenic gamma-delta T-Celllymphomas, T-Cell leukemias, lymphoblastic lymphoma, mantle celllymphoma, marginal zone lymphoma, nasal T-Cell lymphoma, pediatriclymphoma, peripheral T-Cell lymphomas, primary central nervous systemlymphoma, transformed lymphomas, treatment-related T-Cell lymphomas, andWaldenstrom's macroglobulinemia.

Brain cancers include any cancer of the brain tissues. Examples of braincancers include, but are not limited to: gliomas (e.g., glioblastomas,astrocytomas, oligodendrogliomas, ependymomas, and the like),meningiomas, pituitary adenomas, vestibular schwannomas, primitiveneuroectodermal tumors (medulloblastomas), etc.

As used herein, the term “infection” refers to any state in at least onecell of an organism (i.e., a subject) is infected by an infectious agent(e.g., a subject has an intracellular pathogen infection, e.g., achronic intracellular pathogen infection). As used herein, the term“infectious agent” refers to a foreign biological entity (i.e. apathogen) that induces PD-L (PD-L1 and/or PD-L2) expression (e.g.,increased PD-L expression) in at least one cell of the infectedorganism. For example, infectious agents include, but are not limited tobacteria, viruses, protozoans, and fungi. Intracellular pathogens are ofparticular interest. Infectious diseases are disorders caused byinfectious agents. Some infectious agents cause no recognizable symptomsor disease under certain conditions, but have the potential to causesymptoms or disease under changed conditions. The subject methods can beused in the treatment of chronic pathogen infections, for exampleincluding but not limited to viral infections, e.g., retrovirus,lentivirus, hepadna virus, herpes viruses, pox viruses, human papillomaviruses, etc.; intracellular bacterial infections, e.g., Mycobacterium,Chlamydophila, Ehrlichia, Rickettsia, Brucella, Legionella, Francisella,Listeria, Coxiella, Neisseria, Salmonella, Yersinia sp, Helicobacterpylori etc.; and intracellular protozoan pathogens, e.g., Plasmodium sp,Trypanosoma sp., Giardia sp., Toxoplasma sp., Leishmania sp., etc.

Infectious diseases that can be treated using a subject high affinityPD-1 mimic polypeptide include but are not limited to: HIV, Influenza,Herpes, Giardia, Malaria, Leishmania, the pathogenic infection by thevirus Hepatitis (A, B, & C), herpes virus (e.g., VZV, HSV-I, HAV-6,HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus,flaviviruses, echovirus, rhinovirus, coxsackie virus, cornovirus,respiratory syncytial virus, mumps virus, rotavirus, measles virus,rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus,papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus andarboviral encephalitis virus, pathogenic infection by the bacteriachlamydia, rickettsial bacteria, mycobacteria, staphylococci,streptococci, pneumonococci, meningococci and conococci, klebsiella,proteus, serratia, pseudomonas, E. coli, legionella, diphtheria,salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague,leptospirosis, and Lyme's disease bacteria, pathogenic infection by thefungi Candida (albicans, krusei, glabrata, tropicalis, etc.),Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), GenusMucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomycesdermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis andHistoplasma capsulatum, and pathogenic infection by the parasitesEntamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoebasp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii,Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosomacruzi, Leishmania donovani, Toxoplasma gondi, and/or Nippostrongylusbrasiliensis.

In some embodiments, a subject high affinity PD-1 mimic polypeptide canblock the inhibitory signals induced by PD-L (e.g., PD-L1 and/or PD-L2),and thereby allow for the activation of an immune cell. Thus, a subjecthigh affinity PD-1 mimic polypeptide can facilitate and/or stimulatecytokine and/or chemokine production by immune cells, particularlyimmune cells that express PD-1 on the cell surface. For example, thepresence of an immune complex (i.e., an antigen-antibody complex)interacting with an immune cell activates the immune cell and inducescytokine production by the immune cell, but this activation(stimulation) can be inhibited by PD-L on the surface of a second cell.A subject high affinity PD-1 mimic polypeptide can be used for alteringimmunoresponsiveness of an immune cell and thereby may be useful fortreating or preventing an immunological disease or disorder (e.g., adisorder associated with immunosuppression). In other words, a subjecthigh affinity PD-1 mimic polypeptide can be used for immunopotentiation(stimulation of the immune system) as an agent that simulates the immunesystem.

The methods above include administering to an individual in need oftreatment a therapeutically effective amount or an effective dose of asubject high affinity PD-1 mimic polypeptide, including withoutlimitation combinations of a high affinity PD-1 mimic polypeptide with adrug (e.g., a chemotherapeutic drug, a tumor-specific antibody, ananti-inflammatory drug, a drug to treat infection, an immunostimulant,i.e., an immunopotentiator, an agent that simulates the immune system,etc.).

Effective doses of the therapeutic entity of the present disclosure,e.g., for the treatment of cancer, vary depending upon many differentfactors, including means of administration, target site, physiologicalstate of the patient, whether the patient is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic. Usually, the patient is a human, but nonhuman mammals mayalso be treated, e.g., companion animals such as dogs, cats, horses,etc., laboratory mammals such as rabbits, mice, rats, etc., and thelike. Treatment dosages can be titrated to optimize safety and efficacy.

In some embodiments, the therapeutic dosage may range from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 1 mg/kg body weight or 10 mg/kg body weightor within the range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once every two weeks or once a month or once every 3 to 6months. Therapeutic entities of the present disclosure are usuallyadministered on multiple occasions. Intervals between single dosages canbe weekly, monthly or yearly. Intervals can also be irregular asindicated by measuring blood levels of the therapeutic entity in thepatient. Alternatively, therapeutic entities of the present disclosurecan be administered as a sustained release formulation, in which caseless frequent administration is required. Dosage and frequency varydepending on the half-life of the polypeptide in the patient.

In prophylactic applications, a relatively low dosage may beadministered at relatively infrequent intervals over a long period oftime. Some patients continue to receive treatment for the rest of theirlives. In other therapeutic applications, a relatively high dosage atrelatively short intervals is sometimes required until progression ofthe disease is reduced or terminated, and preferably until the patientshows partial or complete amelioration of symptoms of disease.Thereafter, the patent can be administered a prophylactic regime.

In still other embodiments, methods of the present disclosure includetreating, reducing or preventing any of the above discussed conditions,ailments, and/or diseases (e.g., tumor growth, tumor metastasis or tumorinvasion of cancers including lymphomas, leukemias, carcinomas,melanomas, glioblastomas, sarcomas, myelomas, etc.). For prophylacticapplications, pharmaceutical compositions or medicaments areadministered to a patient susceptible to, or otherwise at risk ofdisease in an amount sufficient to eliminate or reduce the risk, lessenthe severity, or delay the outset of the disease, including biochemical,histologic and/or behavioral symptoms of the disease, its complicationsand intermediate pathological phenotypes presenting during developmentof the disease.

Subject high affinity PD-1 mimic polypeptides can be used in vitro andin vivo to monitor the course of disease therapy, for example, bymeasuring the increase or decrease in the number of cells expressingPD-L (PD-L1 and/or PD-L2), particularly chronically infected cellsand/or cancer cells expressing PD-L1, it can be determined whether aparticular therapeutic regimen aimed at ameliorating disease iseffective. For such purposes, high affinity PD-1 mimic polypeptides canbe detectably labeled.

Subject high affinity PD-1 mimic polypeptides can be used in vitro inbinding assays in which they can be utilized in liquid phase or bound toa solid phase carrier. In addition, the polypeptides in theseimmunoassays can be detectably labeled in various ways. Examples oftypes of assays which can utilize high affinity PD-1 mimic polypeptidesare flow cytometry, e.g., FACS, MACS, histochemistry, competitive andnon-competitive immunoassays in either a direct or indirect format; andthe like. Detection of PD-L using a high affinity PD-1 mimic polypeptidecan be done with assays which are run in either the forward, reverse, orsimultaneous modes, including histochemical assays on physiologicalsamples.

Subject high affinity PD-1 mimic polypeptides can be bound to manydifferent carriers and used to detect the presence of PD-L (PD-L1 and/orPD-L2) expressing cells. Examples of well-known carriers include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the disclosure. Those skilled in the art will know ofother suitable carriers for binding proteins, or will be able toascertain such, using routine experimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present disclosure include but are not limited toenzymes, radioisotopes, fluorescent compounds, colloidal metals,nanoparticles, chemiluminescent compounds, and bio-luminescentcompounds. Those of ordinary skill in the art will know of othersuitable labels for binding to the polypeptides of the disclosure, orwill be able to ascertain such, using routine experimentation.Furthermore, the binding of these labels to the polypeptides of thedisclosure can be done using standard techniques common to those ofordinary skill in the art.

PD-L may be detected by subject high affinity PD-1 mimic polypeptideswhen present in biological fluids and tissues. Any sample containing adetectable amount of PD-L can be used. A sample can be a liquid such asurine, saliva, cerebrospinal fluid, blood, serum and the like, or asolid or semi-solid such as tissues, feces, biopsy, and the like, or,alternatively, a solid tissue such as those commonly used inhistological diagnosis.

Another labeling technique which may result in greater sensitivityconsists of coupling the polypeptides to low molecular weight haptens.These haptens can then be specifically detected by means of a secondreaction. For example, it is common to use haptens such as biotin, whichreacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, whichcan react with specific anti-hapten antibodies.

The imaging conjugates of high affinity PD-1 mimic polypeptides can beadministered to the subject in a series of more than one administration.The imaging conjugate compositions may be administered at an appropriatetime before the visualization technique. For example, administrationwithin an hour before direct visual inspection may be appropriate, oradministration within twelve hours before a PET or MRI scan may beappropriate. Care should be taken, however, to not allow too much timeto pass between administration and visualization, as the imagingcompound may eventually be cleared from the patient's system.

Compositions for treatment (e.g., for the treatment of cancer, chronicinfection, immunosuppression, inflammation, etc.) can be administered byparenteral, topical, intravenous, intratumoral, oral, subcutaneous,intraarterial, intracranial, intraperitoneal, intranasal orintramuscular means. A typical route of administration is intravenous orintratumoral, although other routes can be equally effective.

Compositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection can also be prepared. The preparationalso can be emulsified or encapsulated in liposomes or micro particlessuch as polylactide, polyglycolide, or copolymer for enhanced adjuvanteffect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes,Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of thisdisclosure can be administered in the form of a depot injection orimplant preparation which can be formulated in such a manner as topermit a sustained or pulsatile release of the active ingredient. Thepharmaceutical compositions are generally formulated as sterile,substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Toxicity of the high affinity PD-1 mimic polypeptides described hereincan be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., by determining the LD₅₀ (the dose lethalto 50% of the population) or the LD₁₀₀ (the dose lethal to 100% of thepopulation). The dose ratio between toxic and therapeutic effect is thetherapeutic index. The data obtained from these cell culture assays andanimal studies can be used in formulating a dosage range that is nottoxic for use in human. The dosage of the proteins described herein liespreferably within a range of circulating concentrations that include theeffective dose with little or no toxicity. The dosage can vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition.

Also within the scope of the disclosure are kits comprising thecompositions (e.g., high affinity PD-1 mimic polypeptides andformulations thereof) of the disclosure and instructions for use. Thekit can further contain a least one additional reagent, e.g., achemotherapeutic drug, anti-tumor antibody, and anti-infection drug,e.g, an anti-viral drug, etc. Kits typically include a label indicatingthe intended use of the contents of the kit. The term label includes anywriting, or recorded material supplied on or with the kit, or whichotherwise accompanies the kit.

Key to the Sequence Listing

Wild type human PD-1 protein(also known as PDCD1, CD279, PD1, SLEB2, hPD-1, hPD-I, and hSLE1)(bold: transmembrane domain, amino acids 168-191; underline: amino acids  26-147) (SEQ ID NO: 1)MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPLFragment of wild type human PD-1 polypeptide (R87, N91, and R122 are underlined)(example of a subject PD-1 mimic polypeptide; a “wild type fragment PD-1 mimic polypeptide”) (SEQ ID NO: 2)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTER HAC-I PD-1 (High affinity consensus with iso-leucine at position 41) (SEQ ID NO: 3)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDARFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTERHAC-V PD-1 (High affinity consensus with valine at position 41)(SEQ ID NO: 4) DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDARFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTER G2 4-1 (Generation 2, clone 4-1) (SEQ ID NO: 5)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGPDARFRVTQLPNGRDFHLSVVRARRNDSGTYVCGAISLAPKIQIKESLRAELRVTER G2 4-2 (SEQ ID NO: 6)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHIIWHRESPSGQTDTLAAFPEDRSQPGQDARFRVTQLPNGRDFHLSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTER G2 4-3 (SEQ ID NO: 7)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVIWHLESPSGQTDTLAAFPEDRSQPGQDARFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTER G2 4-5 (SEQ ID NO: 8)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSSQTDTLAAFPEDRSQPGPDARFRVTQLPNGRDFQLSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTER G2 4-12 (SEQ ID NO: 9)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHYESPSGQTDTLAAFPEDRSQPGQDARFRVTQLPNGRDFHLSVVRARRNDSGTYVCGIISLAPKIQIKESLRAELRVTER G1 4-12 (SEQ ID NO: 10)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHLNWYRQSPDCKVFKLAAFPEDRSTPNPDCRFRVTQLPNGRDFHMSVVRARRNDSGTYYCGAITISPGPQIKESLRAELRVTER G1 4-2 (SEQ ID NO: 11)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHLIWFRQSPLGQLFKLAAFPEDRSIPRQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYVCGAISYSPEIQIKESLRAELRVTER G1 4-5 (SEQ ID NO: 12)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHLVWFRQSPNGQVRKLAAFPEDRSEPIPDCRFRVTQLPNGRDFHMSVVRARRNDSGTYVCGAISYAAIVQIKESLRAELRVTER G1 4-1 (SEQ ID NO: 13)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFRLVWHRESPGYETDTLASFPEDRSTPLPDCRFRVTQLPNGRDFHMSVVRARRNDSGTYVCGAIAFHPVIQIKESLRAELRVTER G1 4-4 (SEQ ID NO: 14)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFRLVWHRESPNNHAYTLALFPEDRSLPFPDCRFRVTQLPNGRDFHMSVVRARRNDSGTYICGAITFDPRIQIKESLRAELRVTER G1 4-10 (SEQ ID NO: 15)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHLVWHRLSPVYQTVLLAAFPEDRSPPVQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISYDPTIQIKESLRAELRVTER G2 4-10 (SEQ ID NO: 16)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHYDSPSGQTDTLAAFPEDRSQPGPDCRFRITQLPNGRDFHFSVVRARRNDSGTYICGVISLAPKIQIKESLRAELRVTER G2 4-14 (SEQ ID NO: 17)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHYESPSGQTDTLAAFPEDRSQPGPDCRFRVTQLPNGRDFHFSVVRARRNDSGTYICGVISLAPKIQIKESLRAELRVTER G2 4-4 (SEQ ID NO: 18)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHIIWHRESPSCQTDTLAAFPEDRSQPGQDCRFRITQLPNGRDFHFSVVRARRNDSGTFVCGVISLAPKIQIKESLRAELRVTER G2 4-22 (SEQ ID NO: 19)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHIIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRITQLPNGRDFHFSVVRARRNDSGTFVCGVISLAPKIQIKESLRAELRVTER G2 4-6 (SEQ ID NO: 20)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFRLVWHRESPSGQTDTLAAFPEDRSQPGQDCRFRITQLPNGRDFHFSVVRARRNDSGTFVCGAISFAPKIQIKESLRAELRVTER G2 4-7 (SEQ ID NO: 21)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDCRFRITQLPNGRDFHLSVVRARRNDSGTFVCGVISLAPKIQIKESLRAELRVTER G2 4-18 (SEQ ID NO: 22)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVIWHRESPSGQTDTLAAFPEDRSQPGPDCRFRITQLPNGRDFHMSVVRARKNDSGTYVCGIISLAPKIQIKESLRAELRVTER G2 4-23 (SEQ ID NO: 23)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHIIWHRESPSGQTDTLAAFPEDRSQPGPDCRFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTERMultimerized high affinity PD-1 mimic polypeptides(e.g., for improved pharmacokinetics)(i.e.,fusion to a multimerization domain) HAC-V ‘microbody’(HAC-V PD-1 fused to human IgG1 CH3 domain in- cluding hinge region)(SEQ ID NO: 24) DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDARFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTEREPKSCDKTHTCPPCGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK  HAC-V Fc fusion(HAC-V PD-1 fused to human IgG Fc; here human IgG4for reduced effector functions (compared to otherFc regions) and S228P mutation to prevent fab armexchange; AAA linker between PD-1 variant and Fc is included)(SEQ ID NO: 25) DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDARFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTERAAAPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK Fusions to ‘attenuated’ cytokines Example: HAC-IL2 (F42A/D20T)(SEQ ID NO: 39) DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDARFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTERGGGGSGGGGSAPTSSSTKKTQLQLEHLL L TLQMILNGINNYKNPKLTRMLT A KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFL NRWITFCQSIISTLT Fusions to 41BB-agonists Example: HAC-41BBL (SEQ ID NO: 40)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDARFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTERGGGGSGGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQMELRRWAGEGSGSVSLALHLMPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPA Fusions to CD40-agonists Example: HAC-CD40L (SEQ ID NO: 41)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDARFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTERGGGGSGGGGSGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQS1HLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL  Fusions to inhibitors of BTLA and/or CD160Example: HAC-BTLA decoy: (SEQ ID NO: 42)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDARFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTERGGGGSGGGGSWNIHGKESCDVQLYIKRQSEHSILAGDPFELECPVKYCANRPHVTWCKLNGTTCVKLEDRQTSWKEEK NISFFILHFEPVLPNDNGSYRCSANFQSNLIESHSTTLYVTDVKFusions to inhibitors of TIM3 and/or CEACAM1 Example: HAC-TIM3 decoy:(SEQ ID NO: 43) DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDARFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTERGGGGSGGGGSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLK Cysteine mutants (e.g., for PET labeling) HAC-V N91C (SEQ ID NO: 44)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDARFRVTQLPNGRDFHMSVVRARR C DSGTYVCGVISLAPKIQIKESLRAELRVTER  HAC-V R87C (SEQ ID NO: 45)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDARFRVTQLPNGRDFHMSVV C ARRNDSGTYVCGVISLAPKIQIKESLRAELRVTER  HAC-V R122C (SEQ ID NO: 46)DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDARFRVTQLPNGRDFHMSVVRARRNDSGTYVCGV ISLAPKIQIKESLRAELRVTEC  

The invention now being fully described, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade without departing from the spirit or scope of the invention.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

Example 1

The following example demonstrates the creation of high affinity PD-1mimic polypeptides that effectively antagonize the interaction betweenPD-1 and its ligand PD-L1. The high affinity PD-1 mimic polypeptides canbe used as therapeutics for the same indications as PD-1 and PD-L1antibodies (e.g., those that are currently in clinical trials).

FIG. 1A depicts a schematic illustrating PD-L1 on the surface of a tumorcell specifically binding to PD-1 on the surface of a T cell to inhibitactivation of the T cell, thereby allowing the tumor cell to evadedestruction by the immune system. FIG. 1B depicts a schematicillustrating a subject high affinity PD-1 mimic polypeptide specificallybinding to PD-L1 on the surface of a cancer cell, thereby reducing theability of the cancer cell to inhibit T cell activation, which in turnreduces the cancer cell's ability to evade the immune response.

FIG. 2A depicts a structural representation of the interaction of PD-1(upper right) with PD-L1 (lower left). Residues of PD-1 located at thecontact site with PD-L1 are represented as spheres. A PD-1 mimicpolypeptide (comprising wild type amino acid residues) was mutagenizedat the residues that contact PD-L1 to generate a first generationlibrary (Generation 1) of mutated polypeptides, which were displayed onthe surface of yeast cells. Selections based on binding were thenperformed using 100 nM biotinylated human PD-L1. To screen for PD-1mimic polypeptides having even greater affinity for PD-L1, a secondgeneration library (Generation 2) of mutated polypeptides was generated,focusing the mutagenesis on converging positions. 1 nM biotinylatedhuman PD-L1 was used to screen the Generation 2 library. See FIG. 2B forresults from the screens.

The table of FIG. 3 reflects the sequences of the engineered variants(subject high affinity PD-1 mimic polypeptides) that were produced. “G1”variants are from the Generation 1 library while the “G2” variants arefrom Generation 2 library (see FIGS. 2A-2B). Each numbered columnrepresents the amino acid position for each shown residue relative tothe native PD-1 mimic polypeptide set forth as SEQ ID NO: 2 (Thepolypeptide of SEQ ID NO: 2 is a PD-1 mimic polypeptide that includes awild type PD-1 sequence, but lacks a transmembrane domain and lacks thefirst 25 amino acids of wild type PD-1). For each PD-1 mimic polypeptiderecovered, divergence from the wild-type amino acid residue is indicatedwith the single-letter code for the resulting mutation for each variant.The measured Surface Plasmon Resonance (SPR) affinity for PD-L1 isindicated (when measured) at the right. Based on the recoveredsequences, high affinity PD-1 mimic polypeptides were generated thatcontain consensus amino acid mutations, and are referred to as “HAC”(High Affinity Consensus).

FIG. 4 depicts two representative Surface Plasmon Resonance (SPR) plotsfrom binding experiments (for binding to PD-L1) that were performed. Thedissociation half-life for a native PD-1 mimic polypeptide (havingwild-type human PD-1 sequences) was less than one second. By contrast,the dissociation half-life for a high-affinity consensus PD-1 variantHAC-I (a subject high affinity PD-1 mimic polypeptide) was 42.4 minutes,thus demonstrating that the high-affinity PD-1 mimic polypeptide boundwith much higher affinity to PD-L1 than did the native PD-1 mimicpolypeptide.

Experiments were then performed to further test the bindingcharacteristics of some of the produced high-affinity PD-1 mimicpolypeptides compared to a native PD-1 mimic polypeptide. FIGS. 5A-5Cshow that produced high affinity PD-1 mimic polypeptides potently andspecifically bound to PD-L1. Yeast displaying: (FIG. 5A) human PD-L1,(FIG. 5B) human PD-L2, or (FIG. 5C) mouse PD-L1, were stained withlabeled native PD-1 mimic polypeptide streptavidin tetramers (a controlPD-1 mimic polypeptide having wild-type human PD-1 sequences andconjugated to Alexa647). The binding of the labeled native PD-1 mimicpolypeptide to PD-L1 was competed with variable concentrations ofunlabeled high affinity PD-1 mimic polypeptides (concentrationsindicated on the x-axis).

An unlabeled native PD-1 mimic polypeptide (having wild-type human PD-1sequences) antagonized the PD-1/PD-L1 interaction. High-affinity PD-1mimic polypeptides (HAC-V PD-1, G2 4-1, and G2 4-2) potently antagonizedthe PD-1/PD-L1 interaction at much lower concentrations than did thenative PD-1 mimic polypeptide, thus demonstrating that they are in facthigh affinity PD-1 mimic polypeptides (FIG. 1A). The high-affinity PD-1mimic polypeptides did not demonstrate antagonism of the PD-1:PD-L2interaction, while a native PD-1 mimic polypeptide did antagonize thePD-1:PD-L2 interaction. Thus, these particular high-affinity PD-1 mimicpolypeptides had increased affinity for PD-L1 compared to the affinityfor PD-L1 of the native PD-1 mimic polypeptide, but they had decreasedaffinity for PD-L2 compared to the affinity for PD-L2 of the native PD-1mimic polypeptide (FIG. 1B). The produced high-affinity PD-1 mimicpolypeptides were also able to compete for binding to mouse PD-L1 (FIG.5C).

The ability of produced high affinity PD-1 mimic polypeptides toantagonize PD-L1 on human cancer cells was then tested. FIG. 6Ademonstrates that PD-L1 was expressed on the human melanoma cell lineSKMEL28 after induction by stimulation with 2000 U/mL humaninterferon-gamma (IFNγ) for 24 hours (PD-L1 staining was assessed byflow cytometry under induced (plus IFNγ) versus non-induced (minus IFNγ)conditions). IFNγ-stimulated SKMEL28 cells were stained with labelednative PD-1 mimic polypeptide streptavidin tetramers (a control PD-1mimic polypeptide having wild-type human PD-1 sequences and conjugatedto Alexa647) with variable concentrations of unlabeled high-affinityPD-1 mimic polypeptides (concentrations indicated on the x-axis) (FIG.6B). An unlabeled native PD-1 mimic polypeptide (having wild-type humanPD-1 sequences) was ineffective at preventing (required highconcentrations in order to prevent) binding of the labeled native PD-1mimic polypeptide to the SKMEL28 cells (IC50=8.2 μM). By contrast, HAC-V(a high-affinity PD-1 mimic polypeptide) potently inhibited binding ofthe labeled native PD-1 mimic polypeptide (IC50 of 210 pM). HAC-MBH(HAC-V, a high-affinity PD-1 mimic polypeptide, fused to the CH3 domainof human IgG1) inhibited binding of the labeled native PD-1 mimicpolypeptide with additionally enhanced potency (IC50 of 55 pM).

Example 2 Engineering High-Affinity PD-1 Variants for OptimizedImmunotherapy and immunoPET Imaging. (Some Data is Shared with Example1)

Signaling through the immune checkpoint PD-1 enables tumor progressionby dampening anti-tumor immune responses. Therapeutic blockade of thesignaling axis between PD-1 and its ligand PD-L1 with monoclonalantibodies has shown remarkable clinical success in the treatment ofcancer. However, antibodies have inherent limitations that can curtailtheir efficacy in this setting, including poor tissue/tumor penetranceand detrimental Fc-effector functions that deplete immune cells. Todetermine if PD-1/PD-L1 directed immunotherapy could be improved withsmaller, non-antibody therapeutics, directed-evolution by yeast-surfacedisplay was used here to engineer the PD-1 ectodomain as a high-affinity(100 pM) competitive antagonist of PD-L1. In contrast to anti-PD-L1monoclonal antibodies, high-affinity PD-1 demonstrated superior tumorpenetration without inducing depletion of peripheral effector T cells.Consistent with these advantages, in syngeneic CT26 tumor models, highaffinity PD-1 was effective in treating both small (˜50 mm³) and largetumors (>150 mm³), whereas the activity of anti-PD-L1 antibodies wascompletely abrogated against large tumors. Furthermore, high-affinityPD-1 was radiolabeled and applied as a PET imaging tracer to efficientlydistinguish between PD-L1-positive and PD-L1-negative tumors in livingmice, providing an alternative to invasive biopsy and histologicalanalysis. These results highlight the favorable pharmacology of small,non-antibody therapeutics for enhanced cancer immunotherapy and immunediagnostics.

Results

Directed Evolution of High-Affinity PD-1 Variants that Antagonize PD-L1.

Given its modest affinity for PD-L1 (K_(D) of 8.2 μM)¹⁸, the wild-typePD-1 ectodomain is a poor candidate to competitively antagonize thePD-1:PD-L1 interaction in a therapeutic context. The affinity of PD-1for PD-L1 was therefore enhanced using directed evolution withyeast-surface display. The engineering strategy employed a two-libraryapproach. A first library was used to identify mutational “hotspots”that impart large gains in affinity, and a second library served todetermine the optimal combination of beneficial mutations derived fromthe first library.

To design the initial, “first generation” library, the crystal structureof the complex between murine PD-1 (mPD-1) and human PD-L1 (hPD-L1)¹⁹was used to identify 22 corresponding residues in human PD-1 (hPD-1) atthe contact interface with PD-L1 for randomization (FIG. 7A; FIGS.12A-12B). This library was displayed on the surface of the yeast andperformed four rounds of selection using recombinant, biotinylatedhPD-L1 ectodomain as the selection reagent (FIG. 7B, “Generation 1”).Biophysical characterization of the remaining clones showed a 400 to500-fold increase in affinity for hPD-L1, as measured by surface plasmonresonance (FIG. 7C). However, the clones exhibited poor biochemicalbehavior, with decreased expression yield and a tendency towardsaggregation. Inspection of the sequences of the variants (FIG. 7C)showed an average of 16 mutations per clone, with several of therandomized positions converging on a small set of mutations (e.g., V39,N41), while other positions appeared to completely diverge (e.g., S48,D52), or conversely, to have a strict preference for the originalwild-type residue (e.g., P105, E111). The results suggested that the“first generation” variants likely contained a mixture of beneficialmutations, non-functional passenger mutations, and deleteriousmutations, as would be expected given the very large theoreticaldiversity of the library (approximately 10²⁰) that was sampled with 10⁸yeast transformants.

A “second generation” library was thus created to eliminate unnecessaryand deleterious substitutions, while simultaneously optimizingcombinations of mutations that impart enhanced affinity (FIGS. 13A-13B).The library was focused onto those positions that appeared to beconverging away from wild-type and also introduced variation at “core”positions within the PD-1 ectodomain (FIG. 7A). Through 5 rounds ofselection, variants were obtained that strongly bound PD-L1 (FIG. 7B,“Generation 2”). Compared to wild-type hPD-1, the selected variantsbound hPD-L1 with 15,000-40,000 fold enhanced affinity, and showed astrong trend toward convergence onto a consensus sequence of 10 aminoacid substitutions comprising eight contact residues and two coreresidues (FIG. 7C). Two versions of this “high-affinity consensus” (HAC)PD-1 were produced, differing only by an isoleucine or valine atposition 41 (termed HAC-I and HAC-V, respectively), and were found to beindistinguishable by affinity or biochemical behavior. Both HAC-PD-1variants could be easily expressed, were monomeric, and bound hPD-L1with K_(D) values of approximately 100 pM (FIG. 7C). As with the otherhigh-affinity variants, this increase in affinity was largely driven bya dramatic reduction in off-rate, yielding dissociation half-lives ofapproximately 40 minutes, compared to less than one second for thewild-type hPD-1:hPD-L1 interaction (FIG. 8A).

To assess the ability of the engineered PD-1 variants to antagonizePD-L1 on cancer cells, competition binding experiments were performed onhuman and murine melanoma cell lines. On human SK-MEL-28 cells, HAC-Vblocked the binding of wild-type PD-1 tetramers with an IC₅₀ of 210 pM,a 40,000-fold enhancement in potency when compared to wild-type PD-1(IC₅₀=8.2 μM) (FIG. 8B). Though selections were performed using humanPD-L1, HAC-V also showed enhanced blockade of PD-L1 on murine B16-F10cells (IC₅₀=69 nM) compared with wild-type hPD-1 (IC₅₀=2.6 μM), albeitwith a decreased potency relative to its blocking on human cells (FIG.8B). In order to generate a HAC-PD-1 variant that could more efficientlyantagonize mPD-L1 for in vivo studies, the sequence of HAC-V was fusedto the dimeric CH3 domain of human IgG1 to create a HAC “microbody”(HACmb; FIG. 14). By virtue of the increased avidity imparted by itsdimeric structure, HACmb potently blocked both hPD-L1 (IC₅₀=55 pM) andmPD-L1 (IC₅₀=1.2 nM) on SKMEL28 and B16-F10 cells, respectively (FIG.8B). The cross-reactivity of HAC-PD-1 for the second ligand of PD-1,PD-L2, was also characterized. In competition binding experiments onyeast displaying the ectodomain of hPD-L2, HAC-PD-1 did not measurablyinhibit the PD-1:PD-L2 interaction, compared to wild-type PD-1 (IC₅₀=2.5μM; FIG. 8B) Taken in aggregate, these results indicated that HAC-PD-1can potently and specifically antagonize PD-L1 (and could therefore alsoserve as a modular scaffold for further engineering).

Tumor Penetration and T Cell Depletion Studies.

In order to assess PD-L1 binding and tumor penetrance of HAC-PD-1 invivo, genome editing techniques were used to generate sub-lines of themouse colon cancer line CT26 that were either definitively negative formPD-L1 expression, or negative for mPD-L1 but constitutively positivefor hPD-L1 expression. These PD-L1 positive and negative cell linescould be readily distinguished by in vitro staining with eitherfluorescently-labeled anti-hPD-L1 antibody or fluorescently-labeledHAC-PD-1 protein (FIG. 15A). Using these engineered lines, mice wereengrafted bilaterally with PD-L1 negative and hPD-L1 positive tumors.Once the tumors had grown to approximately 1 cm in diameter, a mixtureof fluorophore-labeled anti-hPD-L1 antibody and fluorophore-labeledHAC-PD-1 was systemically delivered by intra-peritoneal injection. After4 hours, the paired tumors were dissected and the degree of binding byeach agent was assessed using both fluorescence microscopy and FACSanalysis.

In all PD-L1 negative tumors, histological analysis revealed nodetectable binding by either anti-PD-L1 antibody or HAC-PD-1, confirmingthe specificity of both agents (FIG. 9A). In contrast, binding of boththe antibody and HAC-PD-1 in hPD-L1 positive tumors were clearlyobserved, but with strikingly different distributions. Whereas theantibody-associated fluorescence signal was limited to peripheralregions of the tumor and cells immediately adjacent to vessels, HAC-PD-1staining was widespread, extending to regions deep within the tumor(FIG. 9A and FIG. 15B). These qualitative observations were supported byFACS analysis of paired PD-L1 positive and negative tumors followingnon-enzymatic dissociation. Neither the antibody nor HAC PD-1 interactedappreciably with the cells of PD-L1 negative tumors (FIG. 9B). However,in hPD-L1-expressing tumors, many cells were positive for HAC-PD-1staining, and a substantial population was positive for both anti-PD-L1antibody and HAC-PD-1 binding (FIG. 9B). In contrast, few if any cellswere positive for anti-PD-L1 antibody staining only (FIG. 9B).Quantification of this signal over multiple experiments revealed asignificant advantage for HAC-PD-1 binding (p<1×10⁻⁴), with more thantwice as many cells on average bound by HAC-PD-1 than by anti-PD-L1antibody (FIG. 9C). Taken together, these data illustrate that HAC-PD-1was able to bind PD-L1 on tumor cells that were otherwise inaccessibleto antibody binding.

In addition to its smaller size, HAC-PD-1 lacks an Fc domain, andtherefore it was reasoned that, in contrast to antibodies, it would notcontribute to an immune-mediated depletion of circulating T cellnumbers. In order to test this hypothesis, wild-type Balb/c mice wereengrafted with tumors derived from the syngeneic colon cancer line CT26,and beginning 14 days post-engraftment, administered daily treatments ofPBS, anti-PD-L1 antibody, or HACmb (used in this case for its enhancedbinding to mPD-L1). 72 hours after initiation of treatment, miceinjected with anti-PD-L1 antibody exhibited a 15% decrease (p=0.011) incirculating peripheral blood CD8+ T cells (FIG. 9D). Though PD-L1expression was detectable on the vast majority of CD4+ and CD8+ cells(FIG. 16), the depletive effect was specific to CD8+ T cells, sparingthe CD4+ compartment (FIG. 9D). In contrast to the antibody, dailytreatment with HACmb protein had no detectable effect on circulating Tcell levels (FIG. 9D), although its effects on lymph node T cells wereslightly more complex. As in the blood, treatment with anti-PD-L1antibody led to a significant depletion of CD8+ T cells (FIG. 9E, ˜20%,p<1×10⁻⁴). However, unlike in the blood, where it had no effect,treatment with HACmb did lead to a slight decrease in CD8+ T cell levelsin the lymph nodes, although to a significantly lesser degree thananti-PD-L1 antibody (FIG. 9E, ˜10%, p=0.022). This observation suggeststhat PD-1/PD-L1-directed agents may have pleiotropic effects on T celldynamics that include Fc-mediated depletion as well as simulation of Tcell trafficking into tumors.

Therapeutic Efficacy of HAC-PD-1 in Syngeneic Tumor Models.

Given that HAC PD-1 agents effectively antagonized both human and mousePD-L1, it was tested whether this blockade could by extension reproducethe anti-tumor effects of anti-PD-L1 antibodies. As an initial test ofthe in vivo efficacy of HAC-PD-1, wild type, immunocompetent Balb/c micewere engrafted with syngeneic CT26 tumors, which have previously beenshown to be responsive to anti-PD-L1 antibodies. On day 7post-engraftment, when tumors had reached approximately 50 mm³ in sizeon average, mice were randomized to treatment cohorts and began dailyinjections with PBS, anti-PD-L1 antibody, or HACmb (FIG. 10A). Asexpected, the tumors of PBS treated mice grew rapidly (FIG. 10B).However, by day 14 treatment with either anti-PD-L1 or HACmbsignificantly slowed tumor growth relative to controls (FIG. 10B,p=2×10⁻⁴ and p<p<1×10⁻⁴, respectively). These two agents displayed anear-identical efficacy in this small tumor study, with no statisticaldifference in tumor growth between the two treatment arms (FIG. 10B,p=0.99). From these in vivo therapeutic results it is concluded that, inthe setting of relatively small tumors, HACmb is indistinguishablyefficacious to well-validated anti-PD-L1 monoclonal antibody treatment.

Many reports of mouse cancer models depend on very early treatment oftumors in order to demonstrate robust therapeutic effects, as per thedesign of the initial experiment. However, given the superior tissuepenetrance of HAC PD-1, and its ability to block PD-1:PD-L1 interactionswithout inducing counterproductive depletion of circulating T cells, itwas hypothesized that its advantages in comparison to antibodies mightbe most apparent when attempting to treat larger, more challengingtumors. To this end, an experiment was initiated in which Balb/c micewere engrafted with CT26 cells, and their tumor volume was monitoreddaily; only when an individual tumor achieved a minimum volume of 150mm³, or roughly three times the average starting size of the previousexperiment, was the host mouse randomized into a cohort and treatmentinitiated. This simple change in experimental protocol had profoundeffects on the comparative efficacy of these agents. Whereas anti-PD-L1antibody and HACmb were equivalent in treating very small CT26 tumors(FIG. 10B), in the case of larger tumors, even daily injection ofanti-PD-L1 antibody failed to register any measurable efficacy over PBStreatment (FIG. 10D, left, p=0.464). In stark contrast, HACmbsignificantly reduced tumor growth in large tumors over the duration ofthe study, as compared to either PBS-treated (FIG. 10D, right, p<1×10⁻⁴)or antibody-treated mice (FIG. 10D, left, p<1×10⁻⁴).

It was next tested whether the superior efficacy of HACmb as amonotherapy would extend in the combination setting (e.g., withanti-CTLA4 antibodies). By itself, anti-CTLA4 antibody therapy waseffective in this large tumor model, slowing the growth of tumorsrelative to PBS treatment (FIG. 10D, left and right, p<1×10⁻⁴); however,co-treatment with anti-PD-L1 antibody alongside anti-CTLA4 antibodyfailed to produce any additional benefit over anti-CTLA4 alone (FIG.10D, left, p=0.756). In contrast, HACmb improved anti-CTLA4 therapy, asmice treated with a combination of anti-CTLA4 and HACmb hadsignificantly smaller tumors as compared to either HACmb (FIG. 10D,p=0.012), or anti-CTLA4 alone (FIG. 10D, p=0.006).

In summary, these in vivo studies demonstrate that HAC PD-1 is effectivein treating syngeneic mouse tumors. Importantly, the results illustratethat increases in tumor size disproportionately affect the efficacy ofanti-PD-L1 antibodies (in fact rendering them ineffective once tumorshave surpassed a certain size threshold), while HAC-PD-1 protein remainsefficacious in a challenging and more clinically realistic tumor model.This observation thus suggests that anti-PD-1 or anti-PD-L1 antibodiesmay not fully capture the maximal therapeutic benefit of PD-1:PD-L1blockade, and that further improvements are possible with optimizedtherapeutic agents.

In Vivo Detection of PD-L1 Expression by Positron Emission Tomography(PET) with ⁶⁴Cu Radiolabeled HAC-PD-1.

Tumor PD-L1 expression has been suggested as a potential biomarker topredict response to PD-1- or PD-L1-directed immunotherapies. At present,PD-L1 expression on tumors is most commonly assessed through biopsyfollowed by immunohistochemical staining. However, in addition to theassociated risk and contraindications of the biopsy procedure, theresulting tissue analysis is complicated by the heterogeneous spatialexpression pattern of PD-L1 within a tumor. “ImmunoPET”-can provide anon-invasive means by which to measure the expression of PD-L1throughout an entire tumor simultaneously, without the need to exciseany tissue. It was reasoned that, owing to its high affinity andspecificity for PD-L1, as well as its enhanced tissue penetration, aradiolabeled HAC-PD1 could thus serve as an effective PET probe toassess tumor PD-L1 expression.

To develop a PET tracer based on the HAC-PD-1 scaffold, a mutatedvariant, HAC-N91C, was conjugated with the thiol-reactive bifunctionalchelate DOTA-maleimide²⁰. While the apparent hPD-L1 affinity of DOTA-HACwas weaker than its parent sequence HAC-V, DOTA-HAC nonethelessantagonized hPD-L1 1,200-fold more potently than WT PD-1 (FIG. 17A).Subsequent radiolabeling with ⁶⁴Cu produced the hPD-L1-specificradio-protein ⁶⁴Cu-DOTA-HAC, which possessed a specific activity of 8-10μCi per μg and radiochemical purity greater than 98% (FIG. 17B). ThisPET tracer was used to visualize whole-body hPD-L1 expression in aliving mouse.

⁶⁴Cu-DOTA-HAC showed a strong tumor/muscle signal (6-fold enhancement,p<0.05) at 1 hour post injection (FIG. 11A, FIG. 18A), with high uptakein the kidney, indicating rapid renal clearance of free drug from blood,and high signal in the liver, consistent with copper-specific binding byliver-expressed proteins (FIG. 18B, FIG. 18E). The lack of signal withinPD-L1 negative tumors (FIG. 11A, FIG. 18C), or in hPD-L1 positive tumorsblocked by prior injection of 500 μg of cold HAC-PD1 (FIGS. 11A-11B)indicated a high degree of specificity of ⁶⁴Cu-DOTA-HAC-PD1 for PD-L1binding. Additional scans were obtained at 2, 4, and 24 hours (FIG. 18A,FIG. 18D), and assessed biodistribution at 24 hours (FIGS. 19A-19B).Maximal tumor uptake was observed at one hour after injection, thoughstrong signal persisted within hPD-L1(+) tumors for at least 24 hours.In sum, the rapid and specific hPD-L1(+) tumor uptake of ⁶⁴Cu-DOTA-HACfacilitates its use as a reagent for clinical imaging applications.

DISCUSSION

Cancer immunotherapy is a treatment paradigm whose remarkabletherapeutic potential is just beginning to be fully realized. Thoughsuccess has been achieved in cancer patients with antibodies targetingthe PD-1:PD-L1 axis, the data provided here show that additionalefficacy can be achieved using an engineered PD-1 receptor decoy,HAC-PD-1. This protein does not share the antibody-inherent limitationsof poor tumor penetration and unwanted depletion of effector T cells.Accordingly, it exerts enhanced anti-tumor activity compared toanti-PD-L1 antibodies towards larger and more established tumors. Theseresults thus highlight the potential of small protein biologics astherapeutics for patients and their broad applicability in modulation ofthe immune system.

In addition to enhanced delivery to tumors, the modular nature of smallproteins like HAC-PD-1 enables facile combination with otherimmunotherapeutics. This is a key consideration in light of the efficacyof combined checkpoint blockade with nivolumab (anti-PD-1) andipilimumab (anti-CTLA4) in melanoma patients²¹ and numerous preclinicalstudies that have demonstrated synergy between antibodies targetingPD-1/PD-L1 and additional immunomodulatory pathways, such as TIM-3²²,LAG-3²³, GITR²⁴, OX-40²⁵, and 4-1BB²⁶. In the case of HAC-PD-1,multi-specific agents targeting synergistic immunomodulatory pathwayscan readily be elaborated by simply fusing multiple small proteinmodules, including other engineered receptor decoys or single-domainantibodies. This design leverages the co-expression of different immunecheckpoint ligands and/or receptors on the same cells to provideenhanced avidity, and thus potency, to the combined agent. Furthermore,multispecific therapeutics simplify treatment regimens by reducing thenumber of separately administered drugs, and by extension, reduce thecosts associated with their separate manufacture and development.

Although generally well tolerated compared to some other cancertreatments, immunomodulatory drugs such as anti-PD-1 and anti-PD-L1antibodies have toxicities that range from mild diarrhea tolife-threating immune-related adverse events, including autoimmunehepatitis, pneumonitis, and colitis^(8,9) Biomarkers and methods toidentify which patients will respond to treatment are urgently needed toavoid unnecessary toxicity in patients who would not otherwise benefitfrom immunotherapy. Tumor PD-L1 expression by immunohistochemistry (IHC)has thus far proven a partial, but imperfect predictor ofanti-PD-1/anti-PD-L1 response²⁷. However, IHC may be an insensitivemeasure of tumor PD-L1 expression and it is conceivable that this methodmay mischaracterize PD-L1 positive tumors as negative. The workpresented here demonstrates that HAC-PD-1 immunoPET imaging of tumorPD-L1 expression can be used as an alternative to immunohistochemistry.This non-invasive approach allows simultaneous imaging of the entiretumor and associated metastases, which may differ from the primary tumorin PD-L1 expression status. Furthermore, PET imaging can be used forrepeat imaging of the same tumor at different time points (e.g., beforeand after treatment), thereby yielding a richer set of diagnosticinformation that would be difficult or impossible to achieve withtraditional biopsy/IHC approaches.

Methods

Mice.

Animal studies were performed in compliance with approval from theAdministrative Panel on Laboratory Animal Care at Stanford University.6-8 week old Balb/c mice, used for syngeneic tumor engraftments andassessment of T cell levels in response to treatments, were obtaineddirectly from The Jackson Laboratory. Nod.Cg-Prkdc.scid.IL2rg.tm1Wjl/SzJ(NSG) mice, used for in vivo assessment of tumor penetrance and PETstudies, were obtained from in-house breeding stocks.

Cell Lines.

The human melanoma cell line SK-MEL-28, the murine melanoma cell lineB16.F10, and murine colon carcinoma cell line CT26 were obtained fromthe ATCC. All cell lines were maintained in a humidified, 5% CO₂incubator at 37° C. SK-MEL-28 cells were subcultured in EMEM medium(ATCC) supplemented with 10% fetal bovine serum (FBS; Thermo FisherScientific). B16.F10 cells were subcultured in DMEM medium (LifeTechnologies) supplemented with 10% FBS and 55 μM 2-mercaptoethanol(Sigma). CT26 cells were grown in RPMI supplemented with 10% FBS.Genetic variants of CT26 were created by simultaneous transduction ofCT26 cells with Cas9-expressing lentivirus, and a lentiviral poolencoding a mixture of two mPD-L1-targeting sgRNAs [sequenceGGCTCCAAAGGACTTGTACG (SEQ ID NO: 56) and GGTCCAGCTCCCGTTCTACA (SEQ IDNO: 57), respectively], designed using the tools atgenome-engineering.org²⁸. At 6 days post-infection, cells were inducedto express high levels of PD-L1 through treatment with 100 ng/mL ofmouse IFNγ, and at 7 days post-infection, cells were harvested andstained with APC-labeled 10F.9G2 antibody. The negative population wassorted, cultured, and the, several days later after recovery of cellnumbers, these cells were subjected to two additional sequential roundsof sorting. This stable negative population was defined asCT26-Δ(mPD-L1). Lentivirus encoding for constitutive, EF1A-drivenexpression of hPD-L1 were generated and used to infect CT26-Δ(mPD-L1)cells in order to generate a human PD-L1 expressing mouse cancer line.These cells were harvested, stained with PE-anti-PD-L1 (clone MIH1,eBioscience), and sorted to purity. This sorting was repeated threetimes in total to generate the engineered sub-lineCT26-Tg(hPD-L1)-Δ(mPD-L1).

Protein Expression and Purification.

The hPD-1 IgV domain (residues 26-147), hPD-L1 IgV and IgC domains(residues 19-239), high-affinity PD-1 variants, and HACmb were assembledas gBlocks by IDT and cloned in-frame into pAcGP67a with acarboxy-terminal 8× histidine tag for secretion from Trichoplusia ni(High Five) cells using baculovirus. The N91C mutation was introducedinto HAC-V using PCR-mediated site-directed mutagenesis. Secretedprotein was purified from conditioned medium by nickel-nitrilotriaceticacid (Ni-NTA) chromatography and desalted into phosphate buffered saline(PBS). Proteins used for functional or in vivo studies in mice wereadditionally subjected to column washes with Triton X-114 to removeendotoxin. Biotinylated proteins were obtained by addition of acarboxy-terminal biotin acceptor peptide sequence (GLNDIFEAQKIEWHE (SEQID NO: 58)) and enzymatic biotinylation with BirA ligase.

Protein Labeling with Amine- and Cysteine-Reactive Probes.

HAC-V N91C was expressed and purified as described above and reduced byapplication of tris(2-carboxyethyl)phosphine (TCEP) to a finalconcentration of 1 mM. The reduced protein was then combined with a20-fold molar excess of AlexaFluor 594 C5 maleimide (Life Technologies),AlexaFluor 647 C2 maleimide (Life Technologies), ormaleimido-mono-amide-DOTA (Macrocyclics) and incubated at roomtemperature for one hour and then 4° C. for an additional 12 hours.Excess free probe was removed by desalting the reaction mixture into PBSusing a VivaSpin protein concentrator (Sartorius Stedim). For DOTA-HAC,reacted protein was exchanged into Hepes buffered saline (HBS; 10 mMHepes pH 7.4, 150 mM NaCl) and concentrated to −5 mg/mL. The number ofchelators coupled per antibody (c/a) was estimated with matrix-assistedlaser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) by comparison of unreacted HAC-N91C and HAC-DOTA.

Low-endotoxin/azide-free Anti-hPD-L1 (clone 29E.2A3, BioLegend) waslabeled with an 8-fold molar excess of AlexaFluor 488 NHS ester for twohours at room temperature (Life Technologies). Free dye was quenched byaddition of TRIS pH 8.0 to a final concentration of 20 mM and thelabeled antibody desalted with a VivaSpin protein concentrator.

Yeast Display and Directed Evolution.

The IgV domains of hPD-1 (residues 26-147), the IgV and IgC domains ofhPD-L1 (residues 19-132), and hPD-L2 (residues 20-123) were displayed onthe surface of S. cerevisiae strain EBY100 as N-terminal fusions to Aga2using the pYAL vector as previously described.

Construction and Selection of the First-Generation hPD-1 Library:

As a crystal structure of hPD-1 complexed to hPD-L1 has yet to bereported, 22 likely contact residues were inferred through the structureof mPD-1 bound to hPD-L1 (PDB ID 3SBW). A library randomizing theseresidues was generated as described by FIGS. 12 s!-12B, using assemblyPCR with primers listed in Table 2. The library had a theoreticaldiversity of approximately 9.5×10¹⁹ unique protein sequences. The PCRproducts were further amplified with primers containing homology to thepYAL vector and co-electroporated together with linearized pYAL intoEBY100 yeast. The resulting library contained 0.9×10⁸ transformants.

Transformed yeast were recovered and expanded in liquid SDCAA medium at30° C. and induced by dilution 1:10 into liquid SGCAA medium andcultured at 20° C. for 24 hours. Appropriate numbers of induced yeastwere used in each round to ensure at least ten-fold coverage of theexpected diversity of the library at each step, and not less than 10⁸cells. All selection steps were carried out at 4° C. using MACS buffer(PBS with 0.5% bovine serum albumin and 2 mM EDTA). Prior to each round,pre-clearing against streptavidin-AlexaFluor 647 (produced in-house) wasperformed with anti-Cy5/Alexa Fluor 647 microbeads (Miltenyi) and an LDMACS column (Miltenyi). For rounds 1-3, positive selection was performedby labeling induced yeast with 1 μM biotinylated hPD-L1 for one hour at4° C., followed secondary staining with streptavidin-AlexaFluor 647, andmagnetic selection with anti-Cy5/AlexaFluor 647 microbeads and an LSMACS column (Miltenyi). For round four, positive selection was performedby staining with 10 nM biotinylated hPD-L1 and secondary labeling withstreptavidin-AlexaFluor 647. Display levels were determined by stainingwith AlexaFluor 488-conjugated anti-cMyc (Cell Signaling Technologies)and the top 1% of display-normalized hPD-L1 binders were isolated usingfluorescence activated cell sorting (FACS) with a FACS Aria cell sorter.After each round of selection, recovered yeast were expanded in SDCAAmedium at 30° C. overnight and later induced at 20° C. by dilution 1:10into SGCAA medium for 24 hours.

Construction and Selection of the Second-Generation hPD-1 Library:

The second generation library was designed to randomize ten contactpositions from the first library that demonstrated convergence away fromthe wild-type residue, as well as seven additional core positions. Thedesign, illustrated by FIG. 13, had a theoretical diversity ofapproximately 9.1×10⁹ unique protein sequences. As for the firstgeneration library, the second-generation library was constructed byassembly PCR with primers listed in Table 3 and co-electroporated withpYAL into EBY100 yeast. The resulting library yielded 1.2×10⁸transformants.

The second-generation library was selected similarly to thefirst-generation library with a few modifications. Round 1-3 wereperformed by staining with 1 μM biotinylated hPD-L1 and magnetic beadselection as described above. For rounds 4 and 5, kinetic selection wasperformed to select for variants with decreased off-rates. Briefly,yeast were stained with 10 nM biotinylated hPD-L1 for one hour at 4° C.After washing with MACS buffer, the yeast were then incubated with 1 μMnon-biotinylated hPD-L1 for six hours at room temperature withagitation. Post-competed yeast were then stained withstreptavidin-AlexaFluor 647 and AlexaFluor 488-conjugated anti-cMyc andthe top 1% of display-normalized binders were isolated by FACS sorting.

Surface Plasmon Resonance.

Experiments were conducted using a Biacore T100 and carried out at 25°C. Biotinylated PD-L1 was immobilized onto a Biacore streptavidin (SA)sensor chip (GE Healthcare) to yield an Rmax of approximately 100 RU. Anunrelated biotinylated protein (the IgSF domain of human CD47) wasimmobilized onto the reference surface with a matching RU value tocontrol for nonspecific binding. Measurements were made with serialdilutions of the PD-1 variants in HBS-P+ buffer (10 mM HEPES pH 7.4, 150mM NaCl, 0.005% surfactant P20) as indicated in FIG. 8A (GE Healthcare).The PD-L1 surface was regenerated by three 60 second injections of 50%v/v ethylene glycol, 100 mM glycine pH 9.5. All data were analyzed withthe Biacore T100 evaluation software version 2.0 with a 1:1 Langmuirbinding model.

PD-1 Cell Competition Binding Assays.

WT PD-1 tetramer was formed by incubating biotinylated WT PD-1 withAlexaFluor 647-conjugated streptavidin at a molar ration of 4:1. PD-L1expression was induced on GFP-luciferase+ SK-MEL-28 cells by overnightsimulation with 2000 U/mL of human IFNγ. 100 nM WT PD-1 tetramer wasthen combined with titrating concentrations of WT PD-1 monomer, HAC-V,or HACmb and simultaneously added to 100,000 induced SK-MEL-28 cells.Cells were incubated with the reagent mixtures on ice for 60 minutesthen washed to remove unbound tetramer. AlexaFluor 647 fluorescenceintensity was quantified by flow cytometry using an Accuri C6 flowcytometer (BD Biosciences).

In Vivo Tumor Penetration Studies.

6-8 week old female Nod.Cg-Prkdc.scid.IL2rg.tm1Wjl/SzJ (NSG) mice wereinjected subcutaneously with 1×10⁶ cells of the genetically modifiedcolon cancer line CT26-Δ(mPD-L1) in their left shoulder, and 1×10⁶ cellsof CT26-Tg(hPD-L1)-Δ(mPD-L1) in their right shoulder, in a 50 μLsuspension of 75% RPMI (Life Technologies) and 25% medium-densitymatrigel (Corning) for each injection. After 14 days, when tumors hadgrown to approximately 1 cm in diameter, mice were injectedintraperitoneally with a mixture of 100 μg AlexaFluor 488-conjugatedanti-PD-L1 antibody (clone 29E.2A3, BioLegend) and 100 μg AlexaFluor594-conjugated HAC PD-1 monomer. After 4 hours, mice were euthanized andtheir tumors were dissected. After several rounds of washing with coldPBS to remove excess blood, each tumor was cut approximately in half.One half was incubated in a solution of 1% PFA in PBS overnight at 4° C.with rocking, washed in PBS, and embedded in Tissue Tek Optimal CuttingTemperature (O.C.T.) (Sakura). 7 micron frozen sections of these tissueswere cut and thawed for 30 minutes, washed in acetone at 4° C. for 4minutes, air-dried for 10 minutes, washed in PBS (three times, 5 minuteseach), and labeled with Hoechst 33342 (Invitrogen) before mounting withFluoromount G (Southern Biotech). Slides were visualized on an Eclipsee800 fluorescent microscope (Nikon) at 10× or 20× magnification. Basicphoto processing, including fluorescence channel false-coloring, channelmerge, and brightness and contrast adjustment, were performed usingAdobe Photoshop (Adobe). For FACS analysis, the second half of eachtumor was finely minced with a straight razor, and the minced tissue waspressed through a 100 μM mesh cell strainer, rinsed with PBS, andfinally passed through a 40 μM cell strainer while in liquid suspension.Samples were kept as close to 4° C. as possible throughout all steps ofprocessing. Finally, the resulting single-cell suspension was fixed in a1% PFA solution, and analyzed for antibody- and HAC-derived fluorescencesignal on an LSRFortessa FACS Analyzer (BD Biosciences).

T Cell Depletion Studies.

6-8 week old wild type female Balb/c mice were shaved on their lowerdorsum and injected subcutaneously with 1×10⁶ cells of the colon cancerline CT26 in a 50 μL suspension of 75% RPMI (Life Technologies) and 25%medium-density matrigel (Corning). Mice whose tumors failed to engraftwithin 7 days by visual inspection were excluded from further study.Those with visible, palpable tumors were randomized into treatmentgroups, 10 mice per group, using the tools at random.org. Mice weretreated for 3 days by once-daily intraperitoneal injections of 100 μlPBS, 250 μg of anti-PD-L1 antibody (clone 10F.9G2, BioXcell), or 250 μgpurified HACmb protein, each adjusted to a concentration of 2.5 mg/mL.After three days of treatment, peripheral blood and lymph nodes werecollected from each mouse and stained with the following panel ofantibodies (BioLegend): AlexaFluor488 CD45 (clone 30-F11), PerCP-Cy5-5CD8 (clone 53-6.7), AlexaFluor700 Nk1.1 (clone PK136), APC-Cy7 B220(clone RA3-6B2), PE-Dazzle CD11b (clone M1/70), PE-Cy5 F4/80 (cloneBM8), PE-Cy7 CD4 (GK1.5), and APC PD-L1 (clone 10F.9G2). DAPI was usedas a viability stain. Samples were analyzed on an LSRFortessa FACSAnalyzer (BD Biosciences).

CT26 Tumor Models.

6-8 week old wild type female Balb/c mice were shaved on their lowerdorsum and injected subcutaneously with 1×10⁶ cells of the colon cancerline CT26 in a 50 μL suspension of 75% RPMI (Life Technologies) and 25%medium-density matrigel (Corning). Mice whose tumors failed to engraftwithin 7 days by visual inspection were excluded from further study. Forsmall tumor treatment studies, mice were randomized into cohorts usingthe list randomization tools at random.org, and treatments wereadministered starting 7 days post-engraftment for all mice. In thesesmall tumor studies, digital caliper measurements were taken every thirdday, and values were graphed as fold change, as normalized to themeasured values on day 10. For large tumor studies, mice were engraftedas described above, and starting at day 8 tumors were measured on adaily basis. Mice were individually sorted into treatment cohorts andtreatment was initiated only when tumors reached a threshold of 150 mm³,approximately 10-14 days post-engraftment in all cases. Digital calipermeasurements were taken every day for every mouse in the large tumorexperiment for the duration of treatment. In order to reduce randomday-to-day variability in measured values, the graphed tumor volumes inthis experiment are averages as evaluated within a sliding window thatincludes the current day, the previous day, and the next day'smeasurements. Values from the large tumor study were graphed as absolutetumor volume (mm³). In both experiments, mice were given daily treatmentinjections intraperitoneally for 14 days with 100 μl PBS, 250 μg ofanti-PD-L1 antibody (clone 10F.9G2, BioXcell), or 250 μg purified HACmbprotein, each adjusted to a concentration of 2.5 mg/mL. Tumors wereapproximated as ellipsoids with two radii x and y, where x is thelargest measurable dimension of the tumor, and y is the dimensionimmediately perpendicular to x: Volume=(4/3)*(π)*(x/2)*(y/2)².

⁶⁴Cu-Labeling of DOTA-HAC.

DOTA-HAC was radiolabeled with ⁶⁴CuCl₂ (University of Wisconsin,Madison): 500 μg of DOTA-HAC in 200 μl of 0.1 mM ammonium acetate buffer(pH 5.5) was reacted with ˜370 MBq (˜10mCi) of neutralized ⁶⁴CuCl₂solution at 37° C. for 1 hour. After incubation, 5 mMethylenediaminetetraacetic acid (EDTA) pH 7.0 was added at roomtemperature for 15 minutes to scavenge unchelated ⁶⁴CuCl₂ in thereaction mixture. Purification of ⁶⁴Cu-DOTA-HAC was performed using anSEC 3000 HPLC with a flow rate of 1.0 mL/min (0.1M phosphate buffer, pH7.0) Radiochemical purity was assessed by radio-HPLC. The final dose ofradioconjugate was passed through a 0.2 μm filter into a sterile vial.

Radiotracer Cell Binding Assay

An in vitro cell binding assay was performed using hPDL1(+) cells,hPDL1(+) cells pre-blocked with HAC-V, and control hPDL1(−) cells toassess immunoreactivitiy. 2.5×10⁵ cells in 0.1 mL were aliquotted intriplicate and washed with PBSA (PBS supplemented with 1% bovine serumalbumin). Each tube was incubated with 0.1 mL, 5 nmol/L ⁶⁴Cu-DOTA-HAC(5-6 MBq/nmol) for 45 minutes. After incubation cells were washed thricewith 1% PBSA. Activity in each cell pellet was quantified using a gammacounter (1470 WIZARD Automatic Gamma Counter; Perkin-Elmer).

Small Animal Micro-PET Imaging.

NSG mice bearing subcutaneous hPDL1 positive (n=4) or hPDL1 negative(n=4) CT26 tumors were injected intravenously with ⁶⁴Cu-DOTA-HAC (˜230μCi/25 μg protein/200 μl PBS). One group also received a blocking dose(n=2) of 500 μg/200 μl cold HAC two hours pre injection of PETradiotracer. Mice were anesthetized and imaged on a Siemens Inveonsmall-animal multimodality PET/CT system (Preclinical Solutions; SiemensHealthcare) at time points of 1, 2, 4, and 24 hours post injection. CTraw images were acquired in the second bed position at 80 kVp/500 pA,half-scan 220° of rotation, 120 projections per bed position, with acone beam micro-X-ray source (50-μm focal spot size) and a 2,048×3,072pixel x-ray detector. CT datasets were reconstructed using a Shepp-Loganfilter and cone-beam filtered back-projection. On the basis ofattenuation correction from the CT scans, static PET images wereacquired with the default coincidence timing window of 3.4 ns and energywindow of 350 t0 650 keV. PET scan acquisition time lengths of 3 minutes(1, 2 hours), 5 minutes (4 hours), and 10 minutes (24 hours) were chosenbased upon time post-injection. PET datasets were reconstructed usingthe two-dimensional ordered-subset expectation maximization (OSEM 2D)algorithm²⁹. Image analysis was performed utilizing the Inveon ResearchWorkspace (IRW). For each microPET scan, three dimensional regions ofinterest (ROI) were drawn over the liver, spleen, kidneys, and tumor ondecay-corrected whole-body images. Percent injected dose per gram oftissue (% ID/g) in each organ was obtained from dividing the mean pixelvalue in the region of interest (ROI; nCi/cc) by the total injecteddose. Partial volume correction was not performed. Statistical analysiswas performed by two-way ANOVA (GraphPad).

Biodistribution Studies.

After completion of micro-PET/CT imaging at the 24 hour post-injectiontime point, mice were euthanized and dissected for biodistribution.Blood and organs (heart, lungs, liver, spleen, pancreas, stomach, smallintestine, large intestine, kidney, muscle, bone, bone marrow, skin,brain, tumor, and tail) were collected and weighed. CPM values for eachorgan from gamma counter measurements were converted to percent-injecteddose per gram of tissue. Data were decay corrected to injection time.

Tables

TABLE 2 Primers used to create “First Generation” PD-1  library SEQ IDPrimer Sequence (5′ to 3′) NO: D1aff_1F CATTTTCAATTAAGATGCAGTTACTTCGCTG59 D1aff_2R AATAACAGAAAATATTGAAAAACAGCGAAGT 60 AACTGCATCTTAATTG D1aff_3FTTACTTCGCTGTTTTTCAATATTTTCTGTTA 61 TTGCTAGCGTTTTAGCAG D1aff_4RGTCTATCTGGGGAATCTGCTAAAACGCTAGC 62 AATAACAGAAAAT D1aff_5FTTTTAGCAGATTCCCCAGATAGACCATGGAA 63 CCCACCAAC D1aff_6RCAACAAAGCTGGGGAGAAAGTTGGTGGGTTC 64 CATGGTC D1aff_7FAACTTTCTCCCCAGCTTTGTTGGTCGTCACT 65 GAAGGTGA D1aff_8RGAACAAGTGAAAGTAGCGTTATCACCTTCAG 66 TGACGACCAA D1aff_9FGTGATAACGCTACTTTCACTTGTTCCTTCTC 67 CAACACTTCC D1aff_10RGAAGGATTCGGAAGTGTTGGAGAAGGAACA 68 D1aff_11FCAACACTTCCGAATCCTTCNDTTTGRWTTGG 69 HWTAGAVWGTCCCCAVNTNDTVWWVYTNDTVNATTGGCTNHTTTCCCAGAAGATAGATCC D1aff_12R GAGTGACTCTGAATCTAGCATCTKGAHNTGG70 TNBGGATCTATCTTCTGGGAAA D1aff_13F AGATGCTAGATTCAGAGTCACTCAATTGCCA 71AAC D1aff_14R GGACATGTGGAAATCTCTACCGTTTGGCAAT 72 TGAGTGACTCTGA D1aff_15FCGGTAGAGATTTCCACATGTCCGTCGTCAGA 73 GCTAGAAGAAACG D1aff_16RGTAAGTACCGGAATCGTTTCTTCTAGCTCTG 74 ACGAC D1aff_17FGAAACGATTCCGGTACTTACNWTTGTGGTGC 75 TATTNCTNDTNHTSCTVNANYTCAAATTAAGVRWTCCTTGAGAGCTGAATTGAG D1aff_18R G GATCCTCTTTCAGTGACTCTCAATTCAGC 76TCTCAAGGA D1aff_19F ATTGAGAGTCACTGAAAGAGGATCCGAACAA 77 AAGCTTATCD1aff_20R CAAGTCTTCTTCGGAGATAAGCTTTTGTTCG 78 GATCCTCTT D1aff_21FAAAGCTTATCTCCGAAGAAGACTTGGGTGGT 79 GGTGG D1aff_22RCCACCAGATCCACCACCACCCAAGTC 80

TABLE 3 Primers used to create “Second Generation” PD-1 library SEQ IDPrimer Sequence (5′ to 3′) NO: 1F_AffMat_G2CATTTTCAATTAAGATGCAGTTACTTCGCTG 81 2R_AffMat_G2AATAACAGAAAATATTGAAAAACAGCGAAGTAACTGC 82 ATCTTAATTG 3F_AffMat_G2TTACTTCGCTGTTTTTCAATATTTTCTGTTATTGCTAG 83 CGTTTTAGCAG 4R_AffMat_G2GTCTATCTGGGGAATCTGCTAAAACGCTAGCAATAA 84 CAGAAAAT 5F_AffMat_G2TTTTAGCAGATTCCCCAGATAGACCATGGAACCCAC 85 CAAC 6R_AffMat_G2CAACAAAGCTGGGGAGAAAGTTGGTGGGTTCCATGG 86 TC 7F_AffMat_G2AACTTTCTCCCCAGCTTTGTTGGTCGTCACTGAAGGT 87 GA 8R_AffMat_G2GAACAAGTGAAAGTAGCGTTATCACCTTCAGTGACG 88 ACCAA 9F_AffMat_G2GTGATAACGCTACTTTCACTTGTTCCTTCTCCAACAC 89 TTCC 10R_AffMat_G2GAAGGATTCGGAAGTGTTGGAGAAGGAACA 90 11F_AffMat_G2CCAACACTTCCGAATCCTTCVRTNTTNWTTGGYWTY 91DTSAWTCCCCATCCDRTCAAACTGATAMATTGGCTG CTTTCCCAGAAG 12R_AffMat_G2GACCTGGTTGGGATCTATCTTCTGGGAAAGCAGCCA 92 AT 13F_AffMat_G2GAAGATAGATCCCAACCAGGTCMAGATGCTAGATTC 93 AGARYTACTCAATTGCCAAACGGTAGAG14R_AffMat_G2 CTTCTAGCTCTGACGACGGASANGTGGAAATCTCTA 94 CCGTTTGGCAATTGAG15F_AffMat_G2 TCCGTCGTCAGAGCTAGAAGAAACGATTCCGGTACT 95 16R_AffMat_G2GCTCTCAAGGATTCCTTAATTTGAANCTTTGGAGCA 96WRGGAAATARYACCACAAANAWRAGTACCGGAATC GTTTCTTCTAGC 17F_AffMat_G2TTCAAATTAAGGAATCCTTGAGAGCTGAATTGAGAGT 97 CAC 18R_AffMat_G2GTTCGGATCCTCTTTCAGTGACTCTCAATTCAGCTCT 98 CAAG 19F_AffMat_G2GTCACTGAAAGAGGATCCGAACAAAAGCTTATCTCC 99 GAAGAAGAC 20R_AffMat_G2CCACCAGATCCACCACCACCCAAGTCTTCTTCGGAG 100 ATAAGCTTTTG

TABLE 4 Statistical analysis of large tumor study groups at treatmentday 14 Mean Individual P Day 14 comparison Diff. 95% CI of diff.Significant? Summary value PBS vs. HACmb 507.6 320.1 to 695.2 Yes ****<0.0001 PBS vs. anti-PD-L1 69.97 −117.6 to 257.5  No ns 0.4636 PBS vs.anti-CTLA4 480.7 293.2 to 668.3 Yes **** <0.0001 PBS vs. anti- 747.4559.9 to 935.0 Yes **** <0.0001 CTLA4 + HACmb PBS vs. anti-CTLA4 + 510.4322.8 to 697.9 Yes **** <0.0001 anti-PD-L1 HACmb vs. anti-PD- −437.7−625.2 to −250.1 Yes **** <0.0001 L1 HACmb vs. anti- −26.92 −214.5 to160.6  No ns 0.7779 CTLA4 HACmb vs. anti- 239.8 52.21 to 427.3 Yes *0.0124 CTLA4 + HACmb HACmb vs. anti- 2.740 −184.8 to 190.3  No ns 0.9771CTLA4 + anti-PD-L1 anti-PD-L1 vs. anti- 410.8 223.2 to 598.3 Yes ****<0.0001 CTLA4 anti-PD-L1 vs. anti- 677.4 489.9 to 865.0 Yes **** <0.0001CTLA4 + HACmb anti-PD-L1 vs. anti- 440.4 252.9 to 628.0 Yes **** <0.0001CTLA4 + anti-PD-L1 anti-CTLA4vs. anti- 266.7 79.13 to 454.2 Yes **0.0055 CTLA4 + HACmb anti-CTLA4vs. anti- 29.66 −157.9 to 217.2  No ns0.7559 CTLA4 + anti-PD-L1 anti-CTLA4 + HACmb −237.0 −424.6 to −49.47Yes * 0.0134 vs. anti-CTLA4 + anti- PD-L1

That which is claimed is:
 1. A high affinity PD-1 mimic polypeptide,wherein the polypeptide is a variant of a wild-type PD1 sequence, which:(a) lacks the PD-1 transmembrane domain, and (b) comprises one or moreamino acid changes relative to a corresponding sequence of the wild typePD-1 polypeptide, wherein the one or more amino acid changes increasesthe affinity of the polypeptide for PD-L1 as compared to the affinityfor PD-L1 of the corresponding wild type PD-1 polypeptide.
 2. The highaffinity PD-1 mimic polypeptide of claim 1, wherein the PD-1 mimicpolypeptide has a K_(d) of 1×10⁻⁷ M or less for PD-L1.
 3. The highaffinity PD-1 mimic polypeptide of claim 1, wherein the one or moreamino acid changes is located at an amino acid position of PD-1 thatcontacts PD-L1.
 4. The high affinity PD-1 mimic polypeptide of claim 3,wherein the one or more amino acid changes is located at an amino acidposition, relative to the PD-1 protein fragment set forth in SEQ ID NO:2, selected from: V39, N41, Y43, M45, S48, N49, Q50, T51, D52, K53, A56,Q63, G65, Q66, L97, S102, L103, A104, P105, K106, and A107; or thecorresponding amino acid position relative to another wild type PD-1protein.
 5. The high affinity PD-1 mimic polypeptide of claim 1, whereinthe one or more amino acid changes is located at an amino acid position,relative to the PD-1 protein fragment set forth in SEQ ID NO: 2,selected from: V39, L40, N41, Y43, R44, M45, S48, N49, Q50, T51, D52,K53, A56, Q63, G65, Q66, V72, H82, M83, R90, Y96, L97, A100, S102, L103,A104, P105, K106, and A107; or the corresponding amino acid positionrelative to another wild type PD-1 protein.
 6. The high affinity PD-1mimic polypeptide of claim 1, wherein the one or more amino acid changesis 5 or more amino acid changes.
 7. The high affinity PD-1 mimicpolypeptide of claim 1, comprising one or more amino acid changes,relative to the PD-1 protein fragment set forth in SEQ ID NO: 2,selected from: (1) V39H or V39R; (2) L40V or L40I; (3) N41I or N41V; (4)Y43F or Y43H; (5) R44Y or R44L; (6) M45Q, M45E, M45L, or M45D; (7) S48D,S48L, S48N, S48G, or S48V; (8) N49C, N49G, N49Y, or N49S; (9) Q50K,Q50E, or Q50H; (10) T51V, T51L, or T51A; (11) D52F, D52R, D52Y, or D52V;(12) K53T or K53L; (13) A56S or A56L; (14) Q63T, Q63I, Q63E, Q63L, orQ63P; (15) G65N, G65R, G65I, G65L, G65F, or G65V; (16) Q66P; (17) V72I;(18) H82Q; (19) M83L or M83F; (20) R90K; (21) Y96F; (22) L97Y, L97V, orL97I; (23) A100I or A100V; (24) S102T or S102A; (25) L103I, L103Y, orL103F; (26) A104S, A104H, or A104D; (27) P105A; (28) K106G, K106E,K106I, K106V, K106R, or K106T; and (29) A107P, A107I, or A107V; or achange that results in the same amino acid at the corresponding positionrelative to another wild type PD-1 protein.
 8. The high affinity PD-1mimic polypeptide of claim 1, comprising amino acid changes located atamino acid positions, relative to the PD-1 protein fragment set forth inSEQ ID NO: 2, selected from: (a) V39, N41, Y43, M45, S48, N49, Q50, K53,A56, Q63, G65, Q66, L97, S102, L103, A104, K106, and A107, or thecorresponding amino acid positions relative to another wild type PD-1protein; (b) V39, N41, Y43, M45, S48, Q50, T51, D52F, K53, A56, Q63,G65, Q66, L97, S102, L103, A104, K106, and A107, or the correspondingamino acid positions relative to another wild type PD-1 protein; (c)V39, L40, N41, Y43, R44, M45, N49, K53, M83, L97, A100, and A107, or thecorresponding amino acid positions relative to another wild type PD-1protein; (d) V39, L40, N41, Y43, M45, N49, K53, Q66P, M83, L97, andA107, or the corresponding amino acid positions relative to another wildtype PD-1 protein; (e) V39, L40, N41, Y43, M45, N49, K53, Q66P, H82,M83, L97, A100, and A107, or the corresponding amino acid positionsrelative to another wild type PD-1 protein; (f) V39, L40, N41, Y43, M45,N49, K53, M83, L97, A100, and A107, or the corresponding amino acidpositions relative to another wild type PD-1 protein; (g) V39, L40, N41,Y43, R44, M45, N49, K53, L97, A100, and A107, or the corresponding aminoacid positions relative to another wild type PD-1 protein; and (h) V39,L40, N41, Y43, M45, N49, K53, L97, A100, and A107, or the correspondingamino acid positions relative to another wild type PD-1 protein.
 9. Thehigh affinity PD-1 mimic polypeptide of any of claim 1, comprising aminoacid changes, relative to the PD-1 protein fragment set forth in SEQ IDNO: 2, selected from: (a) {V39H or V39R}, {N41I or N41V}, {Y43F orY43H}, {M45Q, M45E, M45L, or M45D}, {S48D, S48L, S48N, S48G, or S48V},{N49C, N49G, N49Y, or N49S}, {Q50K, Q50E, or Q50H}, {K53T or K53L},{A56S or A56L}, {Q63T, Q63I, Q63E, Q63L, or Q63P}, {G65N, G65R, G65I,G65L, G65F, or G65V}, {Q66P}, {L97Y, L97V, or L971}, {S102T or S102A},{L103I, L103Y, or L103F}, {A104S, A104H, or A104D}, {K106G, K106E,K106I, K106V, K106R, or K106T}, and {A107P, A107I, or A107V}; or changesthat result in the same amino acids at the corresponding positionsrelative to another wild type PD-1 protein; (b) {V39H or V39R}, {N41I orN41V}, {Y43F or Y43H}, {M45Q, M45E, M45L, or M45D}, {S48D, S48L, S48N,S48G, or S48V}, {Q50K, Q50E, or Q50H}, {T51V, T51L, or T51A}, {D52F,D52R, D52Y, or D52V}, {K53T or K53L}, {A56S or A56L}, {Q63T, Q63I, Q63E,Q63L, or Q63P}, {G65N, G65R, G65I, G65L, G65F, or G65V}, {Q66P}, {L97Y,L97V, or L971}, {S102T or S102A}, {L103I, L103Y, or L103F}, {A104S,A104H, or A104D}, {K106G, K106E, K106I, K106V, K106R, or K106T}, and{A107P, A107I, or A107V}; or changes that result in the same amino acidsat the corresponding positions relative to another wild type PD-1protein; (c) {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43F orY43H}, {R44Y or R44L}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y,or N49S}, {K53T or K53L}, {M83L or M83F}, {L97Y, L97V, or L97I}, {A100Ior A100V}, and {A107P, A107I, or A107V}; or changes that result in thesame amino acids at the corresponding positions relative to another wildtype PD-1 protein; (d) {V39H or V39R}, {L40V or L40I}, {N41I or N41V},{Y43F or Y43H}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, orN49S}, {K53T or K53L}, {Q66P}, {M83L or M83F}, {L97Y, L97V, or L97I},and {A107P, A107I, or A107V}; or changes that result in the same aminoacids at the corresponding positions relative to another wild type PD-1protein; (e) {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43F orY43H}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, or N49S}, {K53Tor K53L}, {Q66P}, {H82Q}, {M83L or M83F}, {L97Y, L97V, or L97I}, {A100Ior A100V}, and {A107P, A107I, or A107V}; or changes that result in thesame amino acids at the corresponding positions relative to another wildtype PD-1 protein; (f) {V39H or V39R}, {L40V or L40I}, {N41I or N41V},{Y43F or Y43H}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, orN49S}, {K53T or K53L}, {M83L or M83F}, {L97Y, L97V, or L97I}, {A100I orA100V}, and {A107P, A107I, or A107V}; or changes that result in the sameamino acids at the corresponding positions relative to another wild typePD-1 protein; (g) {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43For Y43H}, {R44Y or R44L}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G,N49Y, or N49S}, {K53T or K53L}, {L97Y, L97V, or L97I}, {A100I or A100V},and {A107P, A107I, or A107V}; or changes that result in the same aminoacids at the corresponding positions relative to another wild type PD-1protein; and (h) {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43For Y43H}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, or N49S},{K53T or K53L}, {L97Y, L97V, or L97I}, {A100I or A100V}, and {A107P,A107I, or A107V}; or changes that result in the same amino acids at thecorresponding positions relative to another wild type PD-1 protein. 10.The high affinity PD-1 mimic polypeptide of claim 1, comprising theamino acid changes, relative to the PD-1 protein fragment set forth inSEQ ID NO: 2, selected from: (a) V39R, N41V, Y43H, M45E, S48G, N49Y,Q50E, K53T, A56S, Q63T, G65L, Q66P, L97V, S102A, L103F, A104H, K106V,and A107I; or changes that result in the same amino acids at thecorresponding positions relative to another wild type PD-1 protein; (b)V39R, N41V, Y43H, M45E, S48N, Q50H, T51A, D52Y, K53T, A56L, Q63L, G65F,Q66P, L97I, S102T, L103F, A104D, K106R, and A107I; or changes thatresult in the same amino acids at the corresponding positions relativeto another wild type PD-1 protein; (c) V39H, L40V, N41V, Y43H, R44Y,M45E, N49G, K53T, M83L, L97V, A100I, and A107I; or changes that resultin the same amino acids at the corresponding positions relative toanother wild type PD-1 protein; (d) V39H, L40V, N41V, Y43H, M45E, N49G,K53T, Q66P, M83L, L97V, and A107I; or changes that result in the sameamino acids at the corresponding positions relative to another wild typePD-1 protein; (e) V39H, L40V, N41V, Y43H, M45E, N49S, K53T, Q66P, H82Q,M83L, L97V, A100V, and A107I; or changes that result in the same aminoacids at the corresponding positions relative to another wild type PD-1protein; (f) V39H, L40I, N41I, Y43H, M45E, N49G, K53T, M83L, L97V,A100V, and A107I; or changes that result in the same amino acids at thecorresponding positions relative to another wild type PD-1 protein; (g)V39H, L40V, N41I, Y43H, R44L, M45E, N49G, K53T, L97V, A100V, and A107I;or changes that result in the same amino acids at the correspondingpositions relative to another wild type PD-1 protein; (h) V39H, L40V,N41I, Y43H, M45E, N49G, K53T, L97V, A100V, and A107I; or changes thatresult in the same amino acids at the corresponding positions relativeto another wild type PD-1 protein; and (i) V39H, L40V, N41V, Y43H, M45E,N49G, K53T, L97V, A100V, and A107I or changes that result in the sameamino acids at the corresponding positions relative to another wild typePD-1 protein.
 11. The high affinity PD-1 mimic polypeptide of claim 1,wherein the high affinity PD-1 mimic polypeptide comprises a fusionpartner.
 12. The high affinity PD-1 mimic polypeptide of claim 11,wherein the fusion partner is a fragment of a human immunoglobulinpolypeptide sequence.
 13. The high affinity PD-1 mimic polypeptide ofclaim 12, wherein the fragment is selected from: (a) a CH3 domain; and(b) part or whole of an Fc region.
 14. The high affinity PD-1 mimicpolypeptide of claim 11, wherein the fusion partner is selected from: amultimerization domain; a cytokine; an attenuated cytokine; a41BB-agonist; CD40-agonist; an inhibitor of BTLA and/or CD160; and aninhibitor of TIM3 and/or CEACAM1.
 15. The high affinity PD-1 mimicpolypeptide of claim 1, comprising one or more mutations correspondingto R87C, N91C, and/or R122C relative to the PD-1 protein fragment setforth in SEQ ID NO:
 2. 16. The high affinity PD-1 mimic polypeptide ofclaim 1, comprising the amino acid sequence set forth in any of SEQ IDNOs: 3-25, and 39-46.
 17. The high affinity PD-1 mimic polypeptide ofclaim 1, wherein the polypeptide further comprises a detectable label.18. The high affinity PD-1 mimic polypeptide of claim 17, wherein thedetectable label is a positron-emission tomography (PET) imaging label.19. The high affinity PD-1 mimic polypeptide of claim 1, in apharmaceutical formulation.
 20. A nucleic acid comprising a nucleotidesequence encoding the high affinity PD-1 mimic polypeptide of claim 1.21. The nucleic acid according to claim 20, further comprising: (i)nucleotide sequences encoding: (a) a T cell receptor (TCR) alphapolypeptide, and (b) TCR beta polypeptide of a TCR; or (ii) a nucleotidesequence encoding a chimeric antigen receptor (CAR).
 22. A method ofimaging, the method comprising contacting cells expressing PD-L1 with ahigh affinity PD-1 mimic polypeptide as set forth in claim
 17. 23. Amethod of treating an individual having cancer, having a chronicinfection, or having an immunological disorder associated withimmunosuppression, the method comprising: administering to theindividual, a high affinity PD-1 mimic polypeptide according to claim 1,in an amount effective for reducing the binding of PD-1 on a first cellwith PD-L1 on a second cell.